CN115666578A

CN115666578A - Use of complement factor D inhibitors alone or in combination with anti-C5 antibodies for treating paroxysmal nocturnal hemoglobinuria

Info

Publication number: CN115666578A
Application number: CN202180034565.XA
Authority: CN
Inventors: 黄名军; 许雅各; 史蒂文·波多斯; 达拉本·帕特尔; 迈克尔·格夫纳
Original assignee: Alexion Pharmaceuticals Inc
Current assignee: Alexion Pharmaceuticals Inc
Priority date: 2020-05-12
Filing date: 2021-05-11
Publication date: 2023-01-31
Also published as: US20230172930A1; KR20230009431A; AU2021270867A1; CA3178589A1; EP4149473A1; MX2022014275A; WO2021231470A1; JP2023526051A; BR112022022980A2

Abstract

The disclosure provides methods for treating paroxysmal nocturnal hemoglobinuria in a subject who has previously exhibited an inadequate response to anti-C5 antibody therapy by administering to the subject a therapeutically effective amount of an inhibitor of the complement alternative pathway (e.g., an inhibitor that inhibits a target upstream of complement 5 (C5), such as factor D or complement 3 (C3)). The disclosure also provides methods for treating PNH in a human subject comprising administering to the subject a complement factor D inhibitor alone or in combination with an anti-C5 antibody or antigen-binding fragment thereof. In some embodiments, the patient has previously exhibited an inadequate response to anti-C5 antibody therapy.

Description

Use of complement factor D inhibitors alone or in combination with anti-C5 antibodies for treating paroxysmal nocturnal hemoglobinuria

Cross Reference to Related Applications

Priority and benefit of U.S. provisional application No. 63/023,415 (an application filed on 12/5/2020) and U.S. provisional application No. 63/044,431 (an application filed on 26/6/2020), the entire contents of which are incorporated herein by reference, are claimed.

Sequence listing

This application contains a listing of sequences that have been submitted electronically in ASCII format and are incorporated by reference herein in their entirety. The ASCII copy was created at 11/5/2021, named 0618_wo _ _sl. Txt, and was 62,966 bytes in size.

Background

The complement system works in concert with other immune systems of the body to protect against the invasion of cellular and viral pathogens. There are at least 25 complement proteins, which are found to be a complex collection of plasma proteins and membrane cofactors. Plasma proteins account for approximately 10% of the globulins in vertebrate serum. Complement components interact through a series of complex but precise enzymatic cleavage and membrane-bound events, whereby their immune defense functions are achieved. The complement cascade thus elicited results in the production of products with opsonizing, immunomodulating and lytic functions. A brief summary of biological activities associated with complement activation is provided, for example, in The Merck Manual (Merck's Manual of family medical Manual), 16 th edition.

While the normal functioning complement system provides a powerful defense against microbial infection, inappropriate regulation or activation of the complement pathway has been implicated in the pathogenesis of a variety of disorders, including Paroxysmal Nocturnal Hemoglobinuria (PNH). PNH is a disorder primarily caused by intravascular hemolysis and platelet activation, in which uncontrolled complement activity can lead to systemic complications (see socie G et al, french Society of haematology.lancet. [ French Society of hematology-lancets ]1996 (9027): 573-577 and Brodsky, r., blood [ Blood ].2014 (18): 2804-2811. Persistent intravascular hemolysis may be triggered by a variety of stressors, such as infection or physical exertion, and this can lead to increased risk of smooth muscle contraction (free hemoglobin), chronic anemia, and severe thromboembolism. Thromboembolism is the most common cause of death in PNH patients, and the sequelae of such events are pulmonary hypertension and end organ damage to vital organs such as the liver, kidneys, brain, and intestine (Hillmen, p. Et al, am.j. Hematol [ journal of hematology ].2010 (8): 553-559). As a result of these adverse pathological processes, PNH patients have a reduced quality of life (QoL), which may include debilitating fatigue, chronic pain, poor physical function, shortness of breath, abdominal pain, erectile dysfunction, the need for anticoagulation, blood transfusion, and, in some cases, dialysis (Weitz, ic. Et al, thromb Res [ thrombosis study ].2012 (3): 361-368).

PNH patients are at high risk of morbidity and mortality. Accordingly, it is an object of the present disclosure to provide improved methods for treating PNH patients.

Disclosure of Invention

The present disclosure relates in part to the following discoveries: PNH patients who do not respond adequately or fail to respond to anti-C5 antibody therapy benefit from treatment with alternative inhibitors of complement, such as Factor D (FD) inhibitors or C3 inhibitors. In particular, transfusion-dependent PNH patients are on Ekulizumab (eculi)zumab) was also received an oral FD inhibitor (daniscompan) on the basis of eculizumab, showing improved clinical outcome, as shown below: increased levels of hemoglobin (Hgb), improved functional assessment of chronic disease treatment (faciit) fatigue score, reduced transfusion requirements, and improved other PNH parameters. The data show that blocking FD using FD inhibitors (e.g., danipon) is an ongoing use of C5 inhibitors (e.g., using eculizumab)

Ongoing anti-C5 antibody therapy) provides additional benefits to PNH patients. This additional benefit may be due to the prevention of C3-mediated extracellular hemolysis (EVH) and the control of intravascular hemolysis (IVH).

Provided herein are methods for treating Paroxysmal Nocturnal Hemoglobinuria (PNH) in a subject who previously exhibited an inadequate response to a C5 inhibitor (e.g., anti-C5 antibody therapy): administering to the subject a therapeutically effective amount of an inhibitor of an alternative component of an Alternative Pathway (AP). In some embodiments, the inhibitor of the surrogate component of AP is an inhibitor that inhibits a target upstream of complement 5 (C5), such as factor D or complement 3 (C3). In some embodiments, the PNH subject has extravascular hemolysis (EVH).

In some embodiments, the treatment decreases one or more of: (a) persistent extravascular hemolysis (EVH); (b) anemia; and/or (c) transfusion-dependent. In some embodiments, the treatment results in an improvement in the FACIT fatigue scale score. In some embodiments, control of MAC-mediated intravascular hemolysis in an insufficiently responsive PNH subject is maintained or improved after treatment.

In some embodiments, an inadequate response to anti-C5 antibody therapy is associated with: pharmacokinetic (PK) aspects, e.g., (a) ineffective inhibition of C5 cleavage in a subject; (b) Low dose of anti-C5 antibody and/or low subject plasma levels; (C) enhanced clearance of anti-C5 antibodies in the subject; and/or (d) the anti-C5 antibody intolerance in the subject results in a reduction in anti-C5 antibody dose, preferably wherein the anti-C5 antibody intolerance comprises fatigue and post-infusion pain. In some embodiments, an inadequate response to anti-C5 antibody therapy is associated with: pharmacodynamic (PD) aspects, e.g., (a) CR1 polymorphisms; (b) Extravascular hemolysis (EVH), e.g., opsonization of surviving blood cells by intravascular hemolysis (IVH); and/or (C) the deleterious effects of the C3 fragment on anti-C5 antibody activity. In some embodiments, an inadequate response to anti-C5 antibody therapy is associated with one or more PK and PD aspects.

Also provided herein are methods for treating PNH in a human patient, comprising administering to the patient a Complement Factor D (CFD) inhibitor alone or in combination with an anti-C5 antibody or antigen-binding fragment thereof. In some embodiments, the CFD inhibitor and/or the anti-C5 antibody or antigen-binding fragment thereof is administered (or is for administration) according to a particular clinical dosage regimen (e.g., at a particular dose and according to a particular dosing regimen). In some embodiments, the PNH subject has extravascular hemolysis (EVH). In some embodiments, the disclosure relates to methods of treating clinically significant extravascular hemolysis (EVH) in patients (e.g., human patients) with Paroxysmal Nocturnal Hemoglobinuria (PNH). In particular, embodiments of the disclosure relate to treating EVH in a PNH patient who has previously received C5 inhibitor therapy, such as anti-C5 antibody therapy (e.g., therapy with eculizumab or rayleigh mab), the therapy comprising administering to the patient a therapeutically effective amount of a modulator (e.g., an inhibitor) of an alternative component of the complement Alternative Pathway (AP). In some embodiments, the modulator of an alternative component of complement AP comprises an inhibitor of a target upstream of complement 5 (C5), such as an inhibitor of Factor D (FD) or complement 3 (C3); in particular inhibitors of factor D.

In some embodiments of the foregoing or following methods of treatment, the disclosure relates to a method for treating EVH in a PNH patient who has previously received C5 inhibitor therapy (e.g., anti-C5 antibody therapy) comprising administering to the subject a therapeutically effective amount of a factor D inhibitor (e.g., danicopan). In some embodiments, a therapeutically effective amount of the danicopan is administered at a dose of 600mg per day.

In some embodiments of the foregoing or following methods of treatment, the subject is treated withThe above-noted EVH includes (a) anemia (e.g., hgb ≦ 9.5 g/dL) and absolute reticulocyte counts ≧ 120X 10 ⁹ L; and/or (b) at least 1 packed RBC or whole blood infusion within 6 months prior to treatment with an inhibitor of a replacement component of complement AP (e.g., prior to treatment with an FD inhibitor such as dinocap).

In some embodiments of the foregoing or following methods of treatment, administration of an alternative component to complement AP, e.g., a factor D inhibitor such as dannicropan, allows PNH patients with clinically significant EVH to avoid transfusion of blood (TA).

In some embodiments of the foregoing or following methods of treatment, administration of an alternative component of the AP complement pathway (e.g., a factor D inhibitor such as dactinopan) renders PNH patients with clinically significant EVH no longer in need of pRBC transfusion, e.g., PNH patients in need of pRBC transfusion when: (1) Hemoglobin values less than 6g/dL, regardless of the presence or absence of clinical signs or symptoms of PNH; or (2) hemoglobin values less than 9g/dL, with severe signs or symptoms of PNH that require transfusion for relief.

In some embodiments of the foregoing or following methods of treatment, PNH patients with clinically significant EVH are treated with an anti-C5 antibody in combination with a therapeutically effective amount of an inhibitor of an alternative component of AP complement (e.g., eculizumab or rayleigh mab) as an anti-C5 antibody and an FD inhibitor (e.g., daunorubin). In some embodiments, the patient is treated with an FD inhibitor alone (e.g., with dannipagne alone).

In some embodiments of the foregoing or following methods of treatment, a PNH patient with clinically significant EVH is treated with an anti-C5 antibody (e.g., eculizumab) at a standard dose and/or dosing regimen of an anti-C5 antibody in a PNH therapy prior to treatment with an inhibitor of an alternative component of complement AP (e.g., treatment with a factor D inhibitor such as dactinopan)

Or Rayleigh bead monoclonal antibody

) Treatment, and subsequent treatment with the same anti-C5 antibody.

In some embodiments, the disclosure relates to the use of an effective amount of a modulator (e.g., inhibitor) of an alternative component of the complement Alternative Pathway (AP) for treating clinically significant EVH in a patient (e.g., a human patient) having PNH. In particular, embodiments of the present disclosure relate to the use of an effective amount of an inhibitor of a target upstream of C5 (such as an FD or C3 inhibitor; especially an FD inhibitor) to treat EVH in a PNH patient who has previously received treatment with a C5 inhibitor (such as an anti-C5 antibody) (e.g., therapy with eculizumab or rayleigh mab). In particular embodiments, the disclosure relates to the use of an effective amount of danicopan (e.g., an oral dose of 600mg daily) in the treatment of human PNH patients with clinically significant EVH who have been previously treated with eculizumab or rayleigh mab.

In one embodiment, there is provided a method for treating PNH in a subject, the method comprising: administering to the subject a therapeutically effective amount of a Complement Factor D (CFD) inhibitor in combination with a therapeutically effective amount of an anti-C5 antibody or antigen-binding fragment thereof,

wherein the subject exhibits one or more of the following clinical improvements 12 and/or 24 weeks after treatment with the CFD inhibitor:

(a) An increase in hemoglobin of 2.0g/dL or more compared to a baseline hemoglobin level of the subject;

(b) Transfusion independence; and/or

(c) The FACIT fatigue scale score is increased by 10 points or more (e.g., 10, 11, 12) compared to the subject's baseline FACIT fatigue scale score.

In another embodiment, there is provided a method for treating PNH in a subject who previously exhibited an inadequate response to anti-C5 antibody therapy, the method comprising:

administering to the subject a therapeutically effective amount of a Complement Factor D (CFD) inhibitor,

wherein the inadequate response of the subject is transfusion-dependent and/or anemia; and is

(a) An increase in hemoglobin of 2.0g/dL or greater compared to a baseline hemoglobin level of the subject;

(b) Transfusion independence; and/or

administering to the subject a therapeutically effective amount of a Complement Factor D (CFD) inhibitor in combination with a therapeutically effective amount of an anti-C5 antibody or antigen-binding fragment thereof,

wherein the inadequate response of the subject is transfusion-dependent and/or anemia; and

(b) Transfusion independence; and/or

In some embodiments, the methods further comprise determining the hemoglobin level, transfusion status, and/or FACIT fatigue scale score of the subject at baseline and at 12 and/or 24 weeks post-treatment, wherein (a) hemoglobin is increased by 2.0g/dL or more as compared to the subject's baseline hemoglobin level; (b) transfusion independence; and/or (c) an increase in the FACIT fatigue scale score of 10 points or more (e.g., 10, 11, 12) as compared to the subject's baseline FACIT fatigue scale score is indicative of treatment.

In some embodiments, the methods involve treating a subject with PNH who has previously been treated with an anti-C5 antibody therapy (e.g.,

7086 antibody, 8110 antibody, 305LO5 antibody, SKY59 antibody, or REGN3918 antibody) showed an insufficient response. In some embodiments, the subject with PNH was previously paired with

An insufficient response was shown. In some embodiments, a subject with PNH was previously treated at an approved dose or higher without changing the regimen for 8 weeks

Exhibit an inadequate response for ≧ 24 weeks (e.g., 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more weeks). In some embodiments, the subject with PNH was previously paired

An insufficient response was shown.

In some embodiments, the inadequate response of the subject is transfusion-dependent (e.g., ≧ 1 Red Blood Cell (RBC) transfusion at ≦ 12 weeks prior to screening). In some embodiments, the inadequate response of the subject is anemia (e.g., hemoglobin <10 g/dl). In some embodiments, the inadequate response of the subject is transfusion dependence and anemia.

In some embodiments, the subject exhibits one or more clinical improvements after treatment according to the methods described herein. For example, in one embodiment, after treatment, the subject exhibits an increase in hemoglobin of 2.0g/dL or greater compared to the subject's baseline hemoglobin level. In other embodiments, the subject exhibits an increase in hemoglobin of 2.0g/dL or greater after 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 weeks of treatment as compared to the subject's baseline hemoglobin level. In other embodiments, the subject exhibits an increase in hemoglobin of 2.0g/dL or greater compared to the subject's baseline hemoglobin level after 24 weeks of treatment. In other embodiments, after treatment, the subject exhibits transfusion independence. In other embodiments, the subject exhibits transfusion independence after 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 weeks of treatment. In other embodiments, the subject exhibits transfusion independence after 24 weeks of treatment. In other embodiments, after treatment, the subject appears to avoid transfusion. In other embodiments, the subject exhibits avoidance of blood transfusion after 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 weeks of treatment. In other embodiments, the subject exhibits avoidance of blood transfusion after 12 weeks of treatment. In other embodiments, after treatment, the subject exhibits a 10 point or more increase in the FACIT fatigue scale score (e.g., 10, 11, 12) compared to the subject's baseline FACIT fatigue scale score. In other embodiments, after treatment, the subject exhibits an 11 point or more increase in the FACIT fatigue scale score compared to the subject's baseline FACIT fatigue scale score. In other embodiments, the subject exhibits a 10 point or more increase in the FACIT fatigue scale score after 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 weeks of treatment. In other embodiments, the subject exhibits an 11 point or more increase in the FACIT fatigue scale score after 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 weeks of treatment. In other embodiments, the subject exhibits a 10 point or more increase in the FACIT fatigue scale score after 12 weeks of treatment. In other embodiments, the subject exhibits an 11 point or more increase in the FACIT fatigue scale score after 12 weeks of treatment. In other embodiments, the subject exhibits a 10 point or more increase in the FACIT fatigue scale score after 24 weeks of treatment. In other embodiments, the subject exhibits an 11 point or more increase in the FACIT fatigue scale score after 24 weeks of treatment.

In other embodiments, after 12 weeks of treatment, the subject exhibits an increase in hemoglobin compared to the subject's baseline hemoglobin level, and exhibits transfusion independence or avoidance of transfusions. In other embodiments, after treatment (e.g., after 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 weeks), the subject exhibits an increase in hemoglobin of 2.0g/dL compared to the subject's baseline hemoglobin level and exhibits transfusion independence. In other embodiments, after 12 weeks of treatment, the subject exhibits an increase in hemoglobin of 2.0g/dL compared to the subject's baseline hemoglobin level, and exhibits transfusion independence or avoidance of transfusions. In other embodiments, after 24 weeks of treatment, the subject exhibits an increase in hemoglobin of 2.0g/dL compared to the subject's baseline hemoglobin level, and exhibits transfusion independence or avoidance of transfusions.

In other embodiments, after 12 weeks of treatment, the subject exhibits an increase in hemoglobin, and exhibits a 10 point or more increase in the FACIT fatigue scale score, as compared to the subject's baseline hemoglobin level. In other embodiments, after treatment (e.g., after 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 weeks of treatment), the subject exhibits an increase in hemoglobin of 2.0g/dL compared to the subject's baseline hemoglobin level and exhibits an increase in the FACIT fatigue scale score of 10 points or more. In other embodiments, after treatment (e.g., after 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 weeks of treatment), the subject exhibits an increase in hemoglobin of 2.0g/dL compared to the subject's baseline hemoglobin level and exhibits an increase in the FACIT fatigue scale score of 11 points or more. In other embodiments, after 12 weeks of treatment, the subject exhibits an increase in hemoglobin of 2.0g/dL compared to the subject's baseline hemoglobin level and exhibits a 10 point or more increase in the FACIT fatigue scale score. In other embodiments, after 24 weeks of treatment, the subject exhibits an increase in hemoglobin of 2.0g/dL compared to the subject's baseline hemoglobin level and exhibits a 10 point or more increase in the FACIT fatigue scale score. In other embodiments, after 24 weeks of treatment, the subject exhibits an increase in hemoglobin of 2.0g/dL compared to the subject's baseline hemoglobin level and exhibits an increase in the FACIT fatigue scale score of 11 points or more.

In other embodiments, after 12 weeks of treatment, the subject exhibits transfusion independence or avoidance of transfusions and an increase in the FACIT fatigue scale score of 10 points or more. In other embodiments, after treatment (e.g., after 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 weeks of treatment), the subject exhibits transfusion independence or avoidance of transfusions and an increase in the FACIT fatigue scale score of 10 points or more. In other embodiments, after treatment (e.g., after 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 weeks of treatment), the subject exhibits transfusion independence and an increase in the FACIT fatigue scale score of 11 points or more. In other embodiments, the subject exhibits transfusion independence and a 10 point or more increase in the FACIT fatigue scale score after 24 weeks of treatment. In other embodiments, the subject exhibits transfusion independence and an increase in the FACIT fatigue scale score of 11 points or more after 24 weeks of treatment.

In other embodiments, after treatment (e.g., after 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 weeks of treatment), the subject exhibits an increase in hemoglobin of 2.0g/dL compared to the subject's baseline hemoglobin level, and exhibits transfusion independence or avoidance of transfusions and an increase in the FACIT fatigue scale score of 10 points or more. In other embodiments, after treatment (e.g., after 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 weeks of treatment), the subject exhibits an increase in hemoglobin of 2.0g/dL compared to the subject's baseline hemoglobin level, and exhibits transfusion independence or avoidance of transfusions and an increase in the FACIT fatigue scale score of 11 points or more. In other embodiments, after 24 weeks of treatment, the subject exhibits an increase in hemoglobin of 2.0g/dL compared to the subject's baseline hemoglobin level and exhibits an increase in transfusion independent or fatigue scale and FACIT fatigue scale scores of 10 points or more. In other embodiments, after 24 weeks of treatment, the subject exhibits an increase in hemoglobin of 2.0g/dL compared to the subject's baseline hemoglobin level and exhibits a blood transfusion independence or avoidance of blood transfusions and an increase in the FACIT fatigue scale score of 11 points or more.

In some embodiments, the method further comprises determining the hemoglobin level, transfusion status, and/or FACIT fatigue scale score of the subject at baseline and at 12 and/or 24 weeks post-treatment, wherein (a) hemoglobin is increased by 2.0g/dL or more compared to the subject's baseline hemoglobin level; (b) transfusion independence; and/or (c) an increase in the FACIT fatigue scale score of 10 points or more compared to the subject's baseline FACIT fatigue scale score is indicative of treatment.

Any suitable CFD inhibitor can be used in the methods described herein. In some embodiments, the CFD inhibitor is a small molecule inhibitor, nucleotide, peptide, protein, peptidomimetic, aptamer, or any other molecule that binds to factor D. In other embodiments, the CFD inhibitor is a nucleotide selected from the group consisting of: DNA, RNA, shRNA, miRNA, siRNA, and antisense DNA. In other embodiments, the CFD inhibitor is an antibody or antigen-binding fragment thereof that binds to factor D. In other embodiments, the CFD inhibitor comprises:

or a pharmaceutically acceptable salt thereof.

An exemplary CFD inhibitor is danicopan.

Other exemplary CFD inhibitors include compounds 1-7 described by Maibaum, j. Et al (in Nature Chemical Biology, vol. 12, pages 1105-1110 (2016)). Thus, in one embodiment, the CFD inhibitor comprises:

Compound 1

Compound 2

Compound 3 (R = H)

Compound 4 (R = CO) ₂ H)

Compound 5

Compound 6; or

Compound 7.

Another exemplary CFD inhibitor is laplizumab (also known as "FCFD 14S" and "aFD"), as described in WO 2015168468 and U.S. patent No. 10,407,510. Additional exemplary CFD inhibitors include anti-factor D antibodies described in US 20190359699, including mAb 11-8A1, mAb 1F10-5 and variants thereof, the teachings disclosed herein, and specific CFD inhibitors, all of which are expressly incorporated herein by reference.

Other exemplary CFD inhibitors include the fused bicyclic compounds described in U.S. patent No. 6,653,340 (including CFD inhibitor BCX1470 and the compounds disclosed in examples 1-20), and the specific CFD inhibitors described in US 20080269318, including BCX-1470, WO 2012/093101 (see, e.g., US 9,085,555), WO 2014/002057, WO 2014/009833 (see, e.g., US 9,550,755), WO 2014/002051 (see, e.g., US 9,815,819), WO 2014/002052, WO 2014/002053, WO 2014/002054, WO 2014/002058 (see, e.g., US 9,487,483), WO 2014/002059, and WO 2014/005150, wherein the teachings and the CFD inhibitors disclosed therein are all expressly incorporated herein by reference.

Any suitable anti-C5 antibody or antigen-binding fragment thereof can be used in these methods described herein. In some embodiments of the present invention, the, the anti-C5 antibody or antigen-binding fragment thereof is a human antibody, a humanized antibody, a bispecific antibodies, chimeric antibodies, fab'2, scFv, SMIP,

Nanobodies, or domain antibodies.

Exemplary anti-C5 antibodies are

(also known as eculizumab).

Is an anti-C5 antibody comprising heavy and light chains having the sequences shown in SEQ ID NOS 10 and 11, respectively, or antigen-binding fragments and variants thereof. In some embodiments, the anti-C5 antibody comprises a sequence set forth in SEQ ID NO 7

CDR1, CDR2 and CDR3 domains of the VH region, and having the sequence shown in SEQ ID NO. 8

CDR1, CDR2 and CDR3 domains of the VL region. In other embodiments, the antibody comprises heavy chain CDR1, CDR2, and CDR3 domains having sequences set forth in

SEQ ID NOS

1, 2, and 3, respectively, and light chain CDR1, CDR2, and CDR3 domains having sequences set forth in

SEQ ID NOS

4, 5, and 6, respectively. In other embodiments, the antibody comprises VH and VL regions having the amino acid sequences set forth in SEQ ID NO 7 and SEQ ID NO 8, respectively.

Another exemplary anti-C5 antibody is a monoclonal antibody comprising a heavy chain and a light chain having the sequences shown in SEQ ID NOS 14 and 11, respectively

(Rayleigh mab) or antigen binding fragments and variants thereof. In other embodiments, the antibody comprises

The heavy and light chain Complementarity Determining Regions (CDRs) or the Variable Regions (VRs). Thus, in one embodiment, the antibody comprises a sequence set forth in SEQ ID NO 12

CDR1, CDR2 and CDR3 domains of the Variable (VH) heavy chain region, and having the sequence shown in SEQ ID NO. 8

CDR1, CDR2 and CDR3 domains of the light chain Variable (VL) region. In other embodiments, the antibody comprises CDR1, CDR2, and CDR3 heavy chain sequences as set forth in

SEQ ID NOs

19, 18, and 3, and CDR1, CDR2, and CDR3 light chain sequences as set forth in

SEQ ID NOs

4, 5, and 6, respectively. In other embodiments, the antibody comprises VH and VL regions having the amino acid sequences set forth in SEQ ID NO 12 and SEQ ID NO 8, respectively. In other embodiments, the antibody comprises a heavy chain constant region as set forth in SEQ ID NO 13.

In other embodiments, the antibody comprises a variant human Fc constant region that binds to a human neonatal Fc receptor (FcRn), wherein the variant human Fc CH3 constant region comprises Met429Leu and Asn435Ser substitutions at residues corresponding to methionine 428 and asparagine 434 of the native human IgG Fc constant region, each numbered according to EU numbering convention.

In other embodiments, the antibody comprises CDR1, CDR2, and CDR3 heavy chain sequences as set forth in

SEQ ID NOs

19, 18, and 3, respectively, and CDR1, CDR2, and CDR3 light chain sequences as set forth in

SEQ ID NOs

4, 5, and 6, respectively; and a variant human Fc constant region that binds to a human neonatal Fc receptor (FcRn), wherein the variant human Fc CH3 constant region comprises Met429Leu and Asn435Ser substitutions at residues corresponding to methionine 428 and asparagine 434 of a native human IgG Fc constant region, each numbered according to EU numbering convention.

In other embodiments, the anti-C5 antibody comprises the heavy and light chain CDRs or variable regions of the BNJ421 antibody (described in PCT/US2015/019225 and U.S. patent No. 9,079,949). In other embodiments, the anti-C5 antibody comprises the heavy and light chain CDRs or variable regions of the 7086 antibody (see U.S. patent nos. 8,241,628 and 8,883,158). In some embodiments, the anti-C5 antibody or antigen-binding fragment thereof comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences shown in SEQ ID NOs 21, 22, and 23, respectively, and light chain CDR1, CDR2, and CDR3 domains having the sequences shown in SEQ ID NOs 24, 25, and 26, respectively. In some embodiments, the anti-C5 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the sequence shown in SEQ ID No. 27 and a light chain variable region comprising the sequence shown in SEQ ID No. 28.

In some embodiments, the anti-C5 antibody comprises the heavy and light chain CDRs or variable regions of the 8110 antibody (see U.S. patent nos. 8,241,628 and 8,883,158). In some embodiments, the anti-C5 antibody or antigen-binding fragment thereof comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences shown in SEQ ID NOs 29, 30, and 31, respectively, and light chain CDR1, CDR2, and CDR3 domains having the sequences shown in SEQ ID NOs 32, 33, and 34, respectively. In some embodiments, the anti-C5 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the sequence set forth in SEQ ID No. 35 and a light chain variable region comprising the sequence set forth in SEQ ID No. 36.

In some embodiments, the anti-C5 antibody comprises the heavy and light chain CDRs or variable regions of the 305LO5 antibody (see US 2016/0176954 A1). In some embodiments, the anti-C5 antibody or antigen-binding fragment thereof comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences shown in SEQ ID NOs 37, 38, and 39, respectively, and light chain CDR1, CDR2, and CDR3 domains having the sequences shown in SEQ ID NOs 40, 41, and 42, respectively. In some embodiments, the anti-C5 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the sequence shown in SEQ ID No. 43 and a light chain variable region comprising the sequence shown in SEQ ID No. 44.

In some embodiments, the anti-C5 antibody comprises the heavy and light chain CDRs or variable regions of the SKY59 antibody. In some embodiments, the anti-C5 antibody or antigen-binding fragment thereof comprises a heavy chain comprising the sequence shown in SEQ ID NO. 45 and a light chain comprising the sequence shown in SEQ ID NO. 46.

In some embodiments, the anti-C5 antibody comprises the heavy and light chain variable regions or the heavy and light chains of a REGN3918 antibody (see US 20170355757). In some embodiments, the anti-C5 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the sequence set forth in SEQ ID No. 47 and a light chain variable region comprising the sequence set forth in SEQ ID No. 48. In some embodiments, the anti-C5 antibody or antigen-binding fragment thereof comprises a heavy chain sequence set forth in SEQ ID NO. 49 and a light chain sequence set forth in SEQ ID NO. 50.

In other embodiments, the antibody competes with any of the above antibodies for binding to the same epitope on C5 and/or for binding to the same epitope on C5. In other embodiments, the antibody has at least about 90% variable region amino acid sequence identity to any of the above antibodies (e.g., at least about 90%, 95%, or 99% variable region identity to SEQ ID NO:12 or SEQ ID NO: 8).

In other embodiments, the antibody is at pH 7.4 and 25 ℃ with a K ≦ 0.1nM _D Affinity dissociation constant (K) in the range of 1nM or less _D ) Binds to human C5. In other embodiments, the antibody is in K at pH 6.0 and 25 ℃ _D Binding to human C5 at > 10 nM. In yet another embodiment, the [ (antibody or antigen-binding fragment thereof to K of human C5 at pH 6.0 and 25 ℃) of an antibody _D ) /(K of antibody or antigen binding fragment thereof to human C5 at pH 7.4 and 25 deg.C _D )]Greater than 25.

In some embodiments, a CFD inhibitor (e.g., danicopan) is administered (or is used to administer) according to a particular clinical dosage regimen (e.g., at a particular dose and according to a particular dosing regimen). In some embodiments, the CFD inhibitor is administered orally to the subject. In some embodiments, the CFD inhibitor is orally administered to the subject three times daily (TID). In some embodiments, the CFD inhibitor is administered to the subject orally at a dose of about 50mg to 300 mg. In some embodiments, the CFD inhibitor is administered orally at a dose of about 100mg, 110mg, 120mg, 130mg, 140mg, 150mg, 160mg, 170mg, 180mg, 190mg, 200mg, 210mg, 220mg, 230mg, 240mg, 250mg, 260mg, 270mg, 280mg, 290mg, or 300 mg. In some embodiments, the CFD inhibitor is administered orally at a dose of about 100 mg. In some embodiments, the CFD inhibitor is administered orally at a dose TID of about 100 mg. In some embodiments, the CFD inhibitor is administered orally at a dose of about 150 mg. In some embodiments, the CFD inhibitor is administered orally at a dose TID of about 150 mg. In some embodiments, the CFD inhibitor is administered orally at a dose of about 200 mg. In some embodiments, the CFD inhibitor is administered orally at a dose TID of about 200 mg.

In some embodiments, the CFD inhibitor is administered for 4 weeks or more (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 weeks or more). In some embodiments, the CFD inhibitor is administered orally for 24 weeks. In some embodiments, the CFD inhibitor is administered for 9 months, 12 months, 15 months, 20 months, 24 months, or longer. In some embodiments, the CFD inhibitor is administered for 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, or more.

In some embodiments, the anti-C5 antibody or antigen-binding fragment thereof is administered (or used to administer) according to a particular clinical dosage regimen (e.g., at a particular dose and according to a particular dosing regimen) (e.g.,

or

). The anti-C5 antibody or antigen-binding fragment thereof can be administered to the patient by any suitable method. In some embodiments, the anti-C5 antibody or antigen-binding fragment thereof is administered to the subject intravenously.

In some embodiments, the dose of the anti-C5 antibody or antigen-binding fragment thereof is a fixed dose. For example, in some embodiments, the anti-C5 antibody or antigen-binding fragment thereof is administered to the subject at a dose of 600mg per week. In some embodiments, the anti-C5 antibody or antigen-binding fragment thereof is administered to the subject at a dose of 900mg every two weeks.

In some embodiments, the anti-C5 antibody or antigen-binding fragment thereof is administered to the subject at a dose of 600mg per week, four times, followed by 900mg at week 5, and thereafter 900mg every 2 weeks. In some embodiments, will

Administered to a subject (e.g., an adult subject) at a dose of 600mg per week, four times, followed by 900mg at week 5, and thereafter 900mg every 2 weeks.

In some embodiments, the anti-C5 antibody or antigen-binding fragment thereof is administered to a subject younger than 18 years of age and weighing 40kg and above at a dose of 900mg per week, four times, then 1200mg at week 5, and 1200mg every two weeks thereafter. In some embodiments, the method will comprise

Subjects younger than 18 years and weighing 40kg and above were administered four times per week at a dose of 900mg, followed by 1200mg at week 5 and 1200mg every two weeks thereafter.

In some embodiments, the anti-C5 antibody or antigen-binding fragment thereof is administered to a subject less than 18 years old and weighing 30kg to less than 40kg at a dose of 600mg per week, twice per week, followed by 900mg at week 3 And thereafter administered at a dose of 900mg every two weeks. In some embodiments, the method will comprise

Subjects less than 18 years old and weighing 30kg to less than 40kg are administered twice a week at a dose of 600mg, followed by 900mg at week 3 and thereafter 900mg every two weeks.

In some embodiments, the anti-C5 antibody or antigen-binding fragment thereof is administered to a subject less than 18 years of age and weighing 20kg to less than 30kg at a dose of 600mg per week, twice per week, followed by administration at a dose of 600mg at week 3, and thereafter at a dose of 600mg every two weeks. In some embodiments, the method will comprise

Subjects less than 18 years old and weighing 20kg to less than 30kg are administered twice a week at a dose of 600mg, then at week 3 at a dose of 600mg, and thereafter at a dose of 600mg every two weeks.

In some embodiments, the anti-C5 antibody or antigen-binding fragment thereof is administered to a subject less than 18 years old and weighing 10kg to less than 20kg at a dose of 600mg per week, once, then at a dose of 300mg at week 3, and thereafter at a dose of 300mg every two weeks. In some embodiments, the method will comprise

Subjects less than 18 years old and weighing from 10kg to less than 20kg were administered once a week at a dose of 600mg, then at a dose of 300mg on week 3, and then at a dose of 300mg every two weeks thereafter.

In some embodiments, the anti-C5 antibody or antigen-binding fragment thereof is administered to a subject less than 18 years of age and weighing 5kg to less than 10kg at a dose of 300mg per week, once, then at a dose of 300mg at week 2, and thereafter at a dose of 300mg every three weeks. In some embodiments, the method will comprise

Subjects less than 18 years old and weighing 5kg to less than 10kg are administered once a week at a dose of 300mg, followed by administration at a dose of 300mg on week 2, and thereafter at a dose of 300mg every three weeks.

In some embodiments, the dose of the anti-C5 antibody or antigen-binding fragment thereof is based on the weight of the patient. In some embodiments, for example, 300mg of the anti-C5 antibody or antigen-binding fragment thereof is administered to a patient weighing ≧ 5 to <10 kg. In some embodiments, 600mg of the anti-C5 antibody or antigen-binding fragment thereof is administered to a patient weighing ≧ 10 to <20 kg. In some embodiments, 900mg or 2100mg of the anti-C5 antibody or antigen-binding fragment thereof is administered to a patient weighing ≧ 20 to <30 kg. In some embodiments, 1200mg or 2700mg of the anti-C5 antibody or antigen-binding fragment thereof is administered to a patient that weighs ≧ 30 to <40 kg. In some embodiments, 2400mg or 3000mg of an anti-C5 antibody or antigen-binding fragment thereof is administered to a patient weighing ≧ 40 to <60 kg. In some embodiments, 2700mg or 3300mg of the anti-C5 antibody or antigen-binding fragment thereof is administered to a patient weighing ≧ 60 to <100 kg. In some embodiments, 3000mg or 3600mg of the anti-C5 antibody or antigen-binding fragment thereof is administered to a patient weighing ≧ 100 kg. In certain embodiments, the dosage regimen is adjusted to provide the best desired response (e.g., an effective response).

In some embodiments, the anti-C5 antibody or antigen-binding fragment thereof is administered as follows:

(a) Once on day 1 of the administration cycle, the doses were: 2400mg of patients with the weight of more than or equal to 40 to less than 60kg, 2700mg of patients with the weight of more than or equal to 60 to less than 100kg or 3000mg of patients with the weight of more than or equal to 100 kg; and

(b) On day 15 of the administration cycle and every eight weeks thereafter, the doses were: 3000mg for patients with the weight of more than or equal to 40 to less than 60kg, 3300mg for patients with the weight of more than or equal to 60 to less than 100kg, or 3600mg for patients with the weight of more than or equal to 100 kg.

In some embodiments of the present invention, the,

the application was as follows:

In some embodiments, the anti-C5 antibody or antigen-binding fragment thereof is administered to a patient weighing ≧ 40 to <60 kg:

(a) Once on day 1 of the administration cycle, at a dose of 2400mg; and

(b) On day 15 of the administration cycle and every eight weeks thereafter, the doses were: 3000mg.

In some embodiments, the method will comprise

Applied to the body weight of more than or equal to 40<60kg of patients:

(a) Once on day 1 of the administration cycle, at a dose of 2400mg; and

In some embodiments, the anti-C5 antibody or antigen-binding fragment thereof is administered to a patient weighing ≧ 60 to <100 kg:

(a) Once on day 1 of the administration cycle, the dose was 2700mg; and

(b) On day 15 of the administration cycle and every eight weeks thereafter, the doses were: 3300mg.

In some embodiments, the method will comprise

Applied to the body weight of more than or equal to 60 to<100kg of patients:

(a) Once on day 1 of the administration cycle, the dose was 2700mg; and

In some embodiments, the anti-C5 antibody or antigen-binding fragment thereof is administered to a patient weighing ≧ 100 kg:

(a) Once on day 1 of the administration cycle, at a dose of 3000mg; and

(b) On day 15 of the administration cycle and every eight weeks thereafter, the doses were: 3600mg.

In some embodiments, the method will comprise

Applied to patients with the weight more than or equal to 100 kg:

(a) Once on day 1 of the administration cycle, at a dose of 3000mg; and

In some embodiments, the anti-C5 antibody or antigen-binding fragment thereof is administered to a subject less than 18 years of age:

(a) Once on day 1, at the following doses: 600mg of patients with the weight of more than or equal to 5 to less than 10kg, 600mg of patients with the weight of more than or equal to 10 to less than 20kg, 900mg of patients with the weight of more than or equal to 20 to less than 30kg, 1200mg of patients with the weight of more than or equal to 30 to less than 40kg, 2400mg of patients with the weight of more than or equal to 40 to less than 60kg, 2700mg of patients with the weight of more than or equal to 60kg, or 3000mg of patients with the weight of more than or equal to 100 kg; and

(b) On day 15 and every four weeks thereafter, the doses were: 300mg for patients with a body weight of more than or equal to 5 to less than 10kg, or 600mg for patients with a body weight of more than or equal to 10 to less than 20 kg; or on day 15 and every eight weeks thereafter, the doses were: 2100mg of patients with a weight of more than or equal to 20 to less than 30kg, 2700mg of patients with a weight of more than or equal to 30 to less than 40kg, 3000mg of patients with a weight of more than or equal to 40 to less than 60kg, 3300mg of patients with a weight of more than or equal to 60 to less than 100kg, or 3600mg of patients with a weight of more than or equal to 100 kg.

In some embodiments, the anti-C5 antibody or antigen-binding fragment thereof is administered to a body weight ≧ 5 to<10kg of patients: once on day 1 at a dose of 600mg; and (b) on day 15 and every four weeks thereafter at a dose of 300mg. In some embodiments, the method will comprise

Applied to the body weight of more than or equal to 5 to<10kg of patients: once on day 1 at a dose of 600mg; and (b) at day 15 and every four weeks thereafter, at a dose of 300mg.

In some embodiments, the anti-C5 antibody or antigen-binding fragment thereof is administered to a patient weighing ≧ 10 to <20 kg: once on day 1 at a dose of 600mg; and (b) at day 15 and every four weeks thereafter, at a dose of 600mg. In some embodiments, the anti-C5 antibody is administered to a patient weighing ≧ 10 to <20 kg: once on day 1 at a dose of 600mg; and (b) at day 15 and every four weeks thereafter, at a dose of 600mg.

In some embodiments, the anti-C5 antibody or antigen-binding fragment thereof is administered to a body weight ≧ 20 to<30kg of patients: once on day 1, at a dose of 900mg; and (b) on day 15 and every eight weeks thereafter at a dose of 2100mg. In some embodiments, the method will comprise

Applied to the body weight of more than or equal to 20 to<30kg of patients: once on day 1, at a dose of 900mg; and (b) a dose of 2100mg on day 15 and every eight weeks thereafter.

In some embodiments, the anti-C5 antibody or antigen-binding fragment thereof is administered to a body weight ≧ 30<40kg of patients: once on day 1 at a dose of 1200mg; and (b) on day 15 and every eight weeks thereafter, at a dose of 2700mg. In some embodiments, will

Applied to the body weight of more than or equal to 30<40kg of patients: once on day 1 at a dose of 1200mg; and (b) on day 15 and every eight weeks thereafter, at a dose of 2700mg.

In some embodiments, the anti-C5 antibody or antigen-binding fragment thereof is administered to a body weight ≧ 40 to<60kg of patients: once on day 1, dose was 2400mg; and (b) on day 15 and every eight weeks thereafter at a dose of 3000mg. In some embodiments, the method will comprise

Applied to the body weight of more than or equal to 40<60kg of patients: once on day 1, dose was 2400mg; and (b) at day 15 and every eight weeks thereafter, at a dose of 3000mg.

In some embodiments, the anti-C5 antibody or antigen-binding fragment thereof is administered to a body weight ≧ 60 to<100kg of patients: once on day 1, at a dose of 2700mg; and (b) on day 15 and every eight weeks thereafter, at a dose of 3300mg. In some embodiments, the method will comprise

Applied to the body weight of more than or equal to 60 to<100kg of patients: once on day 1, at a dose of 2700mg; and (b) on day 15 and every eight weeks thereafter, at a dose of 3300mg.

In some embodiments, the anti-C5 antibody or antigen-binding fragment thereof is administered to a patient weighing ≧ 100 kg: once on day 1, at a dose of 3000mg; and (b) a dose of 3600mg on day 15 and every eight weeks thereafter. In some embodiments, the method will comprise

Applied to patients with the weight more than or equal to 100 kg: once on day 1, at a dose of 3000mg; and (b) a dose of 3600mg on day 15 and every eight weeks thereafter.

In another aspect, the described treatment regimen is sufficient to maintain a particular serum trough concentration of the anti-C5 antibody or antigen-binding fragment thereof. In one embodiment, for example, the treatment regimen maintains a serum trough concentration of 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 200, 205, 210, 215, 220, 225, 230, 240, 245, 250, 255, 260, 265, 270, 280, 290, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, or 400 μ g/mL or more of the anti-C5 antibody or antigen binding fragment thereof. In some embodiments, the treatment regimen maintains a serum trough concentration of the anti-C5 antibody or antigen-binding fragment thereof of 100 μ g/mL or more, 150 μ g/mL or more, 200 μ g/mL or more, 250 μ g/mL or more, or 300 μ g/mL or more. In some embodiments, the treatment maintains a serum trough concentration of the anti-C5 antibody or antigen-binding fragment thereof between 100 μ g/mL and 200 μ g/mL. In some embodiments, the treatment maintains a serum trough concentration of the anti-C5 antibody or antigen-binding fragment thereof of about 175 μ g/mL.

In some embodiments, to obtain an effective response, the anti-C5 antibody or antigen-binding fragment thereof is administered to the patient in an amount and at a frequency that maintains at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, or 260 μ g of antibody per milliliter of patient's blood. In some embodiments, the anti-C5 antibody is administered to the patient in an amount and at a frequency that maintains between 50 μ g and 250 μ g antibody per milliliter of patient blood. In some embodiments, the anti-C5 antibody is administered to the patient in an amount and at a frequency to maintain 100 μ g to 200 μ g antibody per milliliter of patient blood. In some embodiments, the anti-C5 antibody is administered to the patient in an amount and at a frequency that maintains about 175 μ g of antibody per milliliter of patient blood.

The efficacy of the treatment methods provided herein can be assessed using any suitable means. In some embodiments, treatment shifts bilirubin to normal levels (e.g., from about 0.2-1.2 mg/dL). In some embodiments, the treatment results in a reduction (e.g., a 2, 3, 4, or 5 fold reduction) of reticulocytes compared to baseline. In some embodiments, treatment results in an increase (e.g., 2, 3, 4, or 5 fold increase) in PNH-specific erythrocyte clone size compared to baseline. In some embodiments, treatment results in a reduction (e.g., 2, 3, 4, or 5 fold reduction) of PNH red blood cells opsonized with the C3 fragment as compared to baseline. In some embodiments, the treatment results in a reduced need for transfusion as compared to baseline. In some embodiments, the treatment results in terminal complement inhibition. In some embodiments, the treatment produces at least one therapeutic effect selected from the group consisting of: abdominal pain, dyspnea, dysphagia, chest pain, and erectile dysfunction are reduced or halted compared to baseline. In some embodiments, the treatment shifts to normal levels at least one or more hemolysis-related blood biomarker selected from the group consisting of: free hemoglobin, haptoglobin, reticulocyte count, PNH Red Blood Cell (RBC) clone, and/or D-dimer. In some embodiments, the treatment reduces Major Adverse Vascular Events (MAVEs). In some embodiments, the treatment shifts the estimated glomerular filtration rate (eGFR) or the on-site urine: albumin: creatinine and plasma Brain Natriuretic Peptide (BNP) to normal levels. In some embodiments, the Treatment alters the Quality of Life relative to baseline as assessed by the Quality of Life 30 Core Questionnaire Scale (Quality of Life questonaire-Core 30 Scale) of the 4 th edition and European Cancer Research and Treatment organization (European organization for Research and Treatment of Cancer).

In some embodiments, LDH levels are used to assess responsiveness to therapy (e.g., a decrease in hemolysis assessed by LDH levels indicates an improvement in at least one sign of PNH). In some embodiments, LDH levels in patients treated according to the disclosed methods are reduced to near normal levels or to within 10%, 20%, 30%, 40%, or 50% lower than the values considered normal levels (e.g., within the range of 105-333IU/L (international units/liter)). In some embodiments, the LDH levels of the patients are normalized throughout the treatment maintenance period. In some embodiments, the LDH level of the treated patient is normalized for at least 95% of the time during treatment maintenance. In some embodiments, the LDH level of the treated patient is normalized for at least 90%, 85%, or 80% of the time during treatment maintenance. In some embodiments, prior to initiation of treatment, the patient's LDH level is greater than or equal to 1.5 times the upper normal limit (LDH greater than or equal to 1.5 × ULN).

In one embodiment, a method for use in the past is provided

A method of treating PNH in an inadequately responsive subject (eculizumab), the method comprising:

administering a therapeutically effective to a subjectAn amount of danekang which combines a therapeutically effective amount

(eculizumab) was added to the reaction mixture,

Wherein danicopan is orally administered to the subject at a dose TID of 100mg, 150mg, or 200 mg;

wherein will be

(eculizumab) is administered intravenously to the subject at a dose of 600mg weekly, four times, followed by 900mg at week 5, and thereafter 900mg every 2 weeks; and

(b) Transfusion independence; and/or

(c) The FACIT fatigue scale score is increased by 10 points or more compared to a baseline FACIT fatigue scale score for the subject.

In another embodiment, methods for treating a subject in need thereof are provided

A method of treating PNH in an inadequately responsive subject (eculizumab), the method comprising: administering to a subject a therapeutically effective amount of a danicopan in combination with a therapeutically effective amount of

(eculizumab) was prepared by mixing the following components,

wherein the inadequate response of the subject is transfusion-dependent and/or anemia; and is provided with

Wherein will be

(eculizumab) is administered intravenously to subjects less than 18 years of age:

(a) Administering to a subject weighing 40kg and above four times a week at a dose of 900mg, followed by a dose of 1200mg at week 5, and thereafter at a dose of 1200mg every two weeks;

(b) Administering to a subject weighing 30kg to less than 40kg twice a week at a dose of 600mg, followed by administration at a dose of 900mg at week 3, and thereafter at a dose of 900mg every two weeks;

(c) Administering to a subject weighing 20kg to less than 30kg twice a week at a dose of 600mg, followed by a dose of 600mg at week 3, and thereafter at a dose of 600mg every two weeks;

(d) Administering to a subject weighing 10kg to less than 20kg once a week at a dose of 600mg, followed by a dose of 300mg at week 3, and thereafter at a dose of 300mg every two weeks; or

(e) Administering to a subject weighing 5kg to less than 10kg once a week at a dose of 300mg, followed by a dose of 300mg at week 2, and thereafter at a dose of 300mg every three weeks; and

i. An increase in hemoglobin of 2.0g/dL or greater compared to a baseline hemoglobin level of the subject;

transfusion independent; and/or

increasing the FACIT fatigue scale score by 10 points or more compared to the subject's baseline FACIT fatigue scale score.

In another embodiment, there is provided a method for treating PNH in a subject, the method comprising:

administering to a subject a therapeutically effective amount of a danekang in combination with a therapeutically effective amount

(eculizumab) was added to the reaction mixture,

wherein will be

wherein the subject exhibits one or more of the following

clinical improvements

12 or 24 weeks after treatment with the CFD inhibitor:

(b) Transfusion independence; and/or

(c) The FACIT fatigue scale score is increased by 10 points or more compared to the subject's baseline FACIT fatigue scale score.

In another embodiment, there is provided a method for treating PNH in a subject less than 18 years of age, the method comprising: administering to a subject a therapeutically effective amount of a danekang in combination with a therapeutically effective amount

(eculizumab) was added to the reaction mixture,

wherein dannikopan is orally administered to the subject at a dose TID of 100mg, 150mg or 200 mg;

wherein will be

(eculizumab) intravenous administration:

(a) Administering to a subject weighing 40kg and above four times a week at a dose of 900mg, followed by a dose of 1200mg at week 5, and thereafter a dose of 1200mg every two weeks;

(d) Administering to a subject weighing 10kg to less than 20kg once a week at a dose of 600mg, followed by a dose of 300mg at week 3, and thereafter at a dose of 300mg every two weeks; or alternatively

(e) Administering to a subject weighing 5kg to less than 10kg once a week at a dose of 300mg, followed by a dose of 300mg on week 2, and thereafter at a dose of 300mg every three weeks; and

wherein the subject exhibits one or more of the following clinical improvements 24 weeks after treatment with the CFD inhibitor:

i. An increase in hemoglobin of 2.0g/dL or more compared to a baseline hemoglobin level of the subject;

transfusion independent; and/or

In some embodiments, the methods described herein further comprise determining the subject's hemoglobin level, transfusion status, and/or FACIT fatigue scale score at baseline and at 12 and/or 24 weeks post-treatment, wherein

(b) Transfusion independence; and/or

(c) Increase the FACIT fatigue scale score by 10 points or more compared to the subject's baseline FACIT fatigue scale score

Is indicative of treatment.

In some embodiments, the disclosure relates to a method for treating PNH in a subject who previously exhibited an inadequate response to a C5 inhibitor (e.g., anti-C5 antibody therapy), the method comprising: administering to the subject a therapeutically effective amount of an inhibitor of the complement Alternative Pathway (AP) selected from the group consisting of:

a) MASP-3 inhibitors (e.g., α -MASP-3 monoclonal antibodies (mabs), such as OMS 906);

b) Factor D (FD) inhibitors (e.g., anti-FD mabs such as laplizumab or small molecule FD inhibitors such as dannikopan (ACH-4471) or BCX 9930);

c) Factor B inhibitors (e.g., LNP 023);

d) Compstatin molecules or derivatives thereof (e.g., APL2, APL9, AMY-101);

e) Mini factor H (e.g., mini FH AMY-201); and

f) Factor H fusion proteins (e.g., TT 30).

In some embodiments, the disclosure relates to a method for treating PNH in a subject who previously exhibited an inadequate response to a C5 inhibitor (e.g., anti-C5 antibody therapy), the method comprising: administering to the subject a therapeutically effective amount of danicopan; in particular, wherein a pharmaceutical composition comprising about 100 to about 200mg of dannikopan is administered to a human subject every 8 hours.

In some embodiments, the disclosure relates to a method for treating PNH in a subject who previously exhibited an inadequate response to an anti-C5 inhibitor, the C5 inhibitor selected from the group consisting of:

a) Eculizumab biosimilar (e.g., ABP 959; elizaria; or SB 12);

b) Nomacopan (Nomacopan) (coverin; rVA 576);

c)

(Rayleigh mab);

d) Tedolumab (lfidolumab) (LFG 316);

e) Parze Li Shankang (Pozelimab); and

f) Kovar Li Shankang (Crovalimab) (SKY 059).

Also provided herein are kits for treating PNH. In some embodiments, the kit comprises: (a)A dose of a Complement Factor D (CFD) inhibitor and (b) instructions for using CFD in any of the methods described herein. In some embodiments, the kit comprises: (ii) (a) a dose of a Complement Factor D (CFD) inhibitor; (b) dosage of anti-C5 antibody; and (C) instructions for using the CFD and anti-C5 antibody in any of the methods described herein. In some embodiments, the CFD is danicopan. In some embodiments, the anti-C5 antibody is

Or

Drawings

Figure 1 is a schematic depicting the dosing regimen of a clinical trial.

Figure 2 depicts the history and "on treatment" transfusion data of individual patients in clinical trials. In particular, fig. 2 shows the incidence and units of blood transfusions for each patient 52 weeks prior to initiation of danicopan treatment and during treatment with danicopan.

Fig. 3 is a graph depicting transfusion frequency and unit volume by annualization rate and units.

Fig. 4A-4D are graphs depicting the effect on complement biomarkers and PNH clone size. Specifically, as shown, serum, plasma and whole blood samples were collected on day 1 prior to administration of danicopan (baseline) and at selected time points during the study and were subjected to CP activity (fig. 4A), AP activity by AP hemolysis assay (fig. 4B), plasma Bb concentration (fig. 4C) and PNH granulocyte colony size, PNH red blood cells and C3d ⁺ Measurement of PNH red blood cells (FIG. 4D). Except for C3d using geometric means ⁺ The arithmetic mean and Standard Deviation (SD) of all erythrocytes are shown outside the clone size of PNH erythrocytes and the range for each time point is shown. NHS, normal human serum; BL, baseline; LLN, lower normal limit; ULN, upper normal limit.

Detailed Description

I. Definition of

As used herein, the term "subject" or "patient" is a human patient (e.g., a patient with Paroxysmal Nocturnal Hemoglobinuria (PNH)).

As used herein, the term "pediatric" patient is a human patient under the age of 18 (< 18 years).

PNH is an acquired hemolytic disease, most commonly found in adults (Brodsky R., blood [ Blood ] 126. The disease begins with clonal expansion of hematopoietic stem cells that have acquired somatic mutations in the PIGA gene (Brodsky r., blood [ Blood ]. 124. Thus, PNH blood cells lack the Glycophosphatidylinositol (GPI) anchor protein and lack the membrane-bound complement inhibitory proteins CD55 and CD59. In the absence of CD55, the deposition of complement protein C3 cleavage products on the surface of blood cell membranes is increased, resulting in the cleavage of C5 into C5a and C5b. The pathology and clinical manifestations of PNH patients are caused by uncontrolled terminal complement activation.

C5a is a potent allergenic toxin, chemokine and cell-activating molecule that mediates a variety of proinflammatory and prothrombotic activities (Matis, L and Rollins, S., nat. Med. [ Nature medicine ] 1. C5b recruits terminal complement components C6, C7, C8 and C9 to form the pro-inflammatory, pro-thrombotic, cytolytic pore-forming molecule C5b-9, which normally would be blocked by CD59 on the Red Blood Cell (RBC) membrane. However, in PNH patients, these final steps are performed without examination, ultimately leading to hemolysis and release of free hemoglobin, as well as platelet activation (Hill, a. Et al, blood [ Blood ],121 4985-96,2013. Signs and symptoms of PNH can be attributed to chronic, uncontrolled complement C5 lysis, and C5a and C5b-9 release leads to RBC hemolysis, which together cause:

release of intracellular free hemoglobin and Lactate Dehydrogenase (LDH) into the circulation is a direct result of hemolysis;

hemoglobin irreversibly binds to and inactivates Nitric Oxide (NO) and inhibits NO synthesis;

Vasoconstriction and tissue bed ischemia due to lack of vasodilatory NO, and possible microthrombosis manifested as abdominal pain, dysphagia and erectile dysfunction;

platelet activation; and

pro-inflammatory and pre-thrombotic states.

A significant proportion of PNH patients experience renal insufficiency and pulmonary hypertension (Hillmen, p. Et al, am. J. Hematol. [ journal of american hematology ],85, 553-9,2010[ prospecting for errors found in am. J. Hematol. [ journal of U.S. hematology ], 85. Patients also experience venous or arterial thrombosis in various locations including the abdomen or central nervous system.

In contrast, PNH children often develop nonspecific symptoms associated with underlying bone marrow disease, such as pallor, fatigue, or jaundice, with a low incidence of hemoglobinuria (Ware, r. Et al, n.engl.j.med. [ new england journal of medicine ], 325. Clinical evaluation of pediatric patients also reveals bone marrow failure syndromes such as aplastic anemia and refractory cytopenia (van den Heuvel eibirnk, m., paediatr. Once the bone marrow disease in children is resolved or the PNH clone is enlarged (the cause of which is not clear), the disease eventually progresses to a more typical disease in adults.

As used herein, "anemia" or "anemic" refers to a small number of red blood cells, i.e., hemoglobin <10g/dl.

As used herein, "hemolysis" refers to the disruption or destruction of Red Blood Cells (RBCs). By "intravascular hemolysis" is meant the lysis of RBCs in circulation, thereby releasing hemoglobin into the plasma. The resulting fragment RBC is called a "split cell". By "extravascular hemolysis" is meant the lysis and phagocytosis of RBCs by macrophages in the spleen and liver. Extravascular hemolysis is characterized by spherical red blood cells.

As used herein, "transfusion" refers to the act of transferring blood, blood products, or other liquids into the circulatory system of a subject. A subject who is "transfusion-dependent" is one who has undergone more than or equal to 1 transfusion (e.g., red blood cell transfusion) at a time ≦ 12 weeks prior to screening and/or treatment. A "transfusion independent" subject is a subject that has not received a transfusion (e.g., red blood cell transfusion) for >12 weeks.

As used herein, "avoiding transfusions" means that a subject treated according to the methods described herein remains in a non-transfused state for week 12 of treatment and does not require transfusions. Transfusions (e.g., packed red blood cells (pRBC)) are required when a subject has (1) a hemoglobin value of less than 6g/dL (regardless of the presence or absence of clinical signs or symptoms), or (2) a hemoglobin value of less than 9g/dL, and the signs or symptoms are very severe requiring transfusions. As used herein, "effective treatment" refers to treatment that produces a beneficial effect, e.g., ameliorating at least one symptom of a disease or disorder. The beneficial effect may take the form of an improvement relative to baseline, for example, an improvement relative to measurements or observations made prior to initiation of treatment according to the method. Effective treatment may refer to alleviation of at least one symptom of PNH (e.g., pallor, fatigue, jaundice, anemia, cytopenia, abdominal pain, dyspnea, dysphagia, chest pain, or erectile dysfunction).

The term "effective amount" refers to an amount of an agent that provides the desired biological, therapeutic and/or prophylactic result. The result can be a reduction, amelioration, palliation, alleviation, delay and/or remission of one or more signs, symptoms or causes of disease, or any other desired change in a biological system. In one example, an "effective amount" is an amount of an anti-C5 antibody or antigen-binding fragment thereof that is clinically proven to alleviate at least one symptom of PNH (e.g., pallor, fatigue, jaundice, anemia, cytopenia, abdominal pain, dyspnea, dysphagia, or chest pain). An effective amount may be administered one or more times.

As used herein, the terms "maintenance" and "maintenance phase" are used interchangeably and refer to the second phase of treatment. In certain embodiments, treatment is continued as long as clinical benefit is observed or until uncontrolled toxicity or disease progression occurs.

As used herein, the term "alternative component of complement" refers to a component other than the listed components, e.g., C5, e.g., MASP3, factor D, factor B, C/C5 convertase, and the like.

As used herein, the term "C5 inhibitor" refers in the broadest sense to any molecule that inhibits or antagonizes C5, e.g., an antibody selected from the group consisting of: (a) Eculizumab or a biological analog thereof, such as ABP 959; elizaria; or SB12; (b) Rayleigh mab; (c) tetumumab (LFG 316); (d) Parzer Li Shankang; and (e) Kovar Li Shankang (SKY 059); or C5 protein/peptide inhibitors such as nomamacropin (Coversin; rVA 576).

As used herein, the term "serum trough level" refers to the minimum level of an agent (e.g., an anti-C5 antibody or antigen-binding fragment thereof) or drug present in serum. In contrast, "peak serum level" refers to the highest level of a drug in the serum. "average serum level" refers to the average level of a drug in the serum over time.

Alternative pathway inhibitors

The complement system is activated by three pathways, the Classical Pathway (CP), the Lectin Pathway (LP) and the Alternative Pathway (AP), which converge to a common point, i.e., the activation of the C3 component (see, e.g., ricklin d. Et al, 2010, nat. Immunol. [ natural immunology ] 11. Activation of complement AP is in a constant state of low level activation (commonly referred to as "slow speed operation"). C3 is hydrolyzed in plasma to C3i, which has many of the properties of C3 b. C3i then binds to the plasma protein, factor B. Bound factor B is cleaved by factor D to produce Ba and Bb. Ba is released and the remaining complex consisting of C3iBb forms the alternative pathway C3 convertase. Most of the C3b produced by the invertase is hydrolyzed. However, if C3B comes into contact with the invading microorganism, it will bind and facilitate amplification of the alternative pathway through binding of C3B to factor B. The plasma protein properdin (properdin) stabilizes the C3 convertase to prolong activity. C3b produced in this pathway also produces the C5 convertase C3bBb3b, which results in the production of C5a and C5 b. Notably, C3b produced in CP enters AP to amplify complement activation.

Any suitable inhibitor of complement AP can be used in the methods described herein. In some embodiments, the inhibitor is an inhibitor that inhibits a target upstream of complement 5 (C5). In some embodiments, the inhibitor is a C3 inhibitor. An exemplary C3 inhibitor is APL-2 (pegcetacoplan), a synthetic cyclic peptide conjugated to a polyethylene glycol (PEG) polymer that specifically binds C3 and C3b. Representative inhibitors of the complement pathway are provided in table 1.

Table 1: molecules and targets for PNH therapy are currently being evaluated

(developer/distributor names are provided in brackets)

In some embodiments, the inhibitor is a Complement Factor D (CFD) inhibitor. Complement factor D is a serine protease with only one known natural substrate: factor B binding to C3B (see, volanakis, j.e. et al, 1993, methods in Enzymol [ methods in enzymology ], 223. Factor D has a serum concentration of 2 μ g/ml, being the lowest of any complement proteins (see, e.g., liszewski, m.k. And j.p.atkinson,1993, fundamental Immunology, third edition, w.e.paul. Editor, raven Press, ltd., new york). Factor D cleaves Factor B (FB) to participate in the production of C3 convertase through two steps in the AP cascade: initial C3 convertase (C3 (H2O) Bb) production following spontaneous AP activation in the fluid phase (low speed operation); and the generation of surface-bound C3 convertase (C3 bBb), which mediates significant amplification of the initial activation (amplification loop) and activation of the terminal pathways, leading to opsonization of the target surface by C3b, release of the anaphylatoxins C3a and C5a, and formation of the Membrane Attack Complex (MAC) (see, e.g., yuan et al, haematologica [ hematology ], 3 months 2017; 102 (3): 466-475). Additional regulatory proteins may promote (properdin) or attenuate (factor H, factor I, various membrane bound proteins, including CD55 and CD 59) AP activity.

As used herein, a "factor D inhibitor" or "CFD inhibitor" is a molecule or substance that prevents, reduces, or blocks the activity of factor D. In some embodiments, the CFD inhibitor is an antibody or antigen-binding fragment thereof, e.g., an antibody or antigen-binding fragment thereof that binds factor D. In some embodiments, the CFD inhibitor is a small molecule inhibitor. In some embodiments, the CFD inhibitor is a nucleotide (e.g., DNA, RNA, shRNA, miRNA, siRNA, or antisense DNA). In some embodiments, the CFD inhibitor is a peptide, protein, peptidomimetic, aptamer, or any other molecule that binds to factor D.

One exemplary CFD inhibitor is danicopan (also known as "ALXN2040", "ACH-4471", and "ACH-0144471"). Dannikopan is a selective and orally active small molecule factor D inhibitor with high binding affinity for human factor D and a Kd of 0.54nM. Danicopan inhibits complement AP (APC) activity. In one embodiment, the CFD inhibitor comprises:

or a pharmaceutically acceptable salt thereof.

Additional exemplary CFD inhibitors include small molecule CFD inhibitors taught by Maibaum, j, et al (Nature Chemical Biology, vol. 12, pages 1105-1110 (2016)), i.e., compounds 1-7, or pharmaceutically acceptable salts thereof. Thus, in one embodiment, the CFD inhibitor comprises:

Compound 1

Compound 2

Compound 3 (R = H)

Compound 4 (R = CO) ₂ H)

Compound 5

Compound 6; or alternatively

Compound 7.

Another exemplary CFD inhibitor is laplizumab (also referred to as "FCFD 4S" and "aFD"), which is an antigen binding fragment of a humanized monoclonal antibody that binds to complement factor D. Specifically, laplizumab is an antibody Fab fragment consisting of a 214 residue light chain (SEQ ID NO: 51) and a 223 residue heavy chain (SEQ ID NO: 52). Laplizumab is described in WO 2015168468 and U.S. patent No. 10,407,510, the teachings of which are expressly incorporated herein by reference. Additional exemplary CFD inhibitors include anti-factor D antibodies described in US 20190359699, including mAb 11-8A1, mAb 1F10-5, and variants thereof (see, e.g., paragraphs [007] - [0021 ]), the teachings of which are expressly incorporated herein by reference.

Other exemplary CFD inhibitors include fused bicyclic compounds described in U.S. patent No. 6,653,340 (see, e.g., column 6 (line 15) to column 56 (line 48)), (including CFD inhibitor BCX 1470) and the compounds disclosed in examples 1-20), as well as specific CFD inhibitors described in US 20080269318, including BCX-1470 (see, e.g., paragraphs [0023] and [0032 ]), WO 2012/093101 (see, e.g., pages 5-67), WO 2014/002057 (see, e.g., pages 3-13), WO 2014/009833 (e.g., pages 4-11), WO 2014/002051 (see, e.g., pages 5-16), WO 2014/002052 (see, e.g., pages 4-11), WO 2014/0023 (see, e.g., pages 4-11), WO 2014/002054 (see, e.g., pages 5-002052), 2014/002052 (see, pages 4-058), WO 2014/002053 (see, pages 3, e.g., pages 3, and WO 2014-00220), and specific CFD inhibitors described herein incorporated by reference.

The CFD inhibitors described herein may be administered, for example, systemically or locally. Systemic administration includes, for example, oral administration, transdermal administration, subcutaneous administration, intraperitoneal administration, subcutaneous administration, nasal administration, sublingual administration, or rectal administration. For example, local administration includes local administration. In one embodiment, the CFD inhibitor is danicopan administered orally.

In some embodiments, the inhibitor of the alternative complement component is a complement 3 (C3) inhibitor, which can be used to reduce the effects of suboptimal C5 blockade (e.g., persistent anemia). It is speculated that the reduction in hematological benefit brought by anti-C5 antibodies in many PNH patients may be due to opsonization of the C3 fragment to viable PNH red blood cells, thereby reducing the half-life of the red blood cells in vivo. Thus, in certain embodiments, the methods of the present disclosure relate to the use of a C3 inhibitor (alone or with an FD inhibitor) in the treatment of PNH patients who are insufficiently responsive to a C5 inhibitor, e.g., anti-C5 antibody therapy with eculizumab.

In some embodiments, the C3 inhibitor is compstatin or an analog thereof. Compstatin is a cyclic peptide that binds to C3 and inhibits complement activation (see U.S. Pat. No. 6,319,897). Compstatin analogs have been developed with a complement inhibitory activity greater than that of compstatin, e.g., WO 2004/026328 (see, e.g., US 7,989,589). As used herein, the term "compstatin analog" includes complement inhibiting analogs of compstatin. See WO2017/062879; WO/2014/152391 and WO/2012/178083, the disclosures of these publications and their U.S. counterparts (see, e.g., US 2019-0381129, US 10,308,687; and US 10,039,802) are incorporated herein by reference in their entirety. Preferred compstatin analogs include pegcetacoplan (APL-2) and related molecules (e.g., APL-9). The compstatin analog may be acetylated or amidated, for example, at the N-terminus and/or C-terminus, particularly at the N-terminus and amidated at the C-terminus.

In some embodiments, the C3 inhibitor is a compstatin mimetic. Representative compstatin mimetics are provided in WO 2004/026328, WO2007/062249, WO/2008/140637, WO/2015/142701, the disclosures of these publications and their U.S. counterparts (see, e.g., US 7,989,589, US 7,888,323, US2011-0046075; and US 10,213,476) are incorporated herein by reference in their entirety. Preferred compstatin mimetics include AMY-101 (CAS: 1427001-89-5).

In some embodiments, the alternative complement component modulates factor H. Factor H regulates complement activation on cells and surfaces by having the cofactor activity of factor I-mediated C3b cleavage and decay-accelerating activity against the alternative pathway C3 convertase C3 bBb. Factor H protects cells and their surfaces, but not the surfaces of bacteria or viruses. This is believed to be a result of the ability of factor H to adopt a conformation with lower or higher activity as a cofactor for C3 cleavage or decay accelerating activity. In a preferred embodiment, the complement component is Mini factor H (Mini FH/AMY-201).

anti-C5 antibodies

The anti-C5 antibodies described herein bind complement component C5 (e.g., human C5) and inhibit cleavage of C5 into fragments C5a and C5b. anti-C5 antibodies (or VH/VL domains derived therefrom) suitable for use in the present disclosure can be produced using methods known in the art. Alternatively, art-recognized anti-C5 antibodies may be used. Antibodies or any other agent that competes with these art-recognized antibodies for binding to C5 may also be used.

The term "antibody"describes polypeptides comprising at least one antibody-derived antigen-binding site (e.g., a VH/VL region or Fv, or CDR). Antibodies include known antibody formats. For example, the antibody can be a human antibody, a humanized antibody, a bispecific antibody, or a chimeric antibody. The antibody may be Fab, fab'2, scFv, SMIP, or,

Nanobodies or domain antibodies. The antibody may also be of any of the following isotypes: igG1, igG2, igG3, igG4, igM, igA1, igA2, igAsec, igD and IgE. The antibody may be a naturally occurring antibody or may be an antibody that has been altered by protein engineering techniques (e.g., by mutation, deletion, substitution, conjugation to a non-antibody moiety). For example, an antibody can include one or more variant amino acids that alter a property (e.g., a functional property) of the antibody (as compared to a naturally occurring antibody). For example, many such changes are known in the art that affect, for example, half-life, effector function, and/or the patient's immune response to an antibody. The term antibody also includes artificial or engineered polypeptide constructs comprising at least one antibody-derived antigen-binding site.

Ekulizumab (also known as

) Is an anti-C5 antibody comprising heavy and light chains having the sequences shown in SEQ ID NOS 10 and 11, respectively, or antigen-binding fragments and variants thereof.

The variable regions of (A) are described in PCT/US1995/005688 and U.S. patent No. 6,355,245, the teachings of which are incorporated herein by reference.

The full heavy and light chains of (a) are described in PCT/US2007/006606 (see, e.g., US 9,718,880), the teachings of which are incorporated herein by reference. In one embodiment, the anti-C5 antibody comprises a sequence shown in SEQ ID NO. 7

CDR1, CDR2 and CDR3 domains of the VL region. In another embodiment, the antibody comprises heavy chain CDR1, CDR2, and CDR3 domains having sequences shown in SEQ ID NOS: 1, 2, and 3, respectively, and light chain CDR1, CDR2, and CDR3 domains having sequences shown in SEQ ID NOS: 4, 5, and 6, respectively. In another embodiment, the antibody comprises VH and VL regions having the amino acid sequences set forth in SEQ ID NO 7 and SEQ ID NO 8, respectively.

Another exemplary anti-C5 antibody is a heavy and light chain comprising the sequences set forth in SEQ ID NOS 14 and 11, respectively

(Rayleigh mab) or antigen binding fragments and variants thereof.

(Rayleigh mab) (also known as BNJ441 and ALXN 1210) is described in PCT/US2015/019225 and U.S. Pat. No. 9,079,949, the teachings of which are incorporated herein by reference. Throughout this document, the terms rayleigh mab, BNJ441 and ALXN1210 are used interchangeably, but all refer to the same antibody.

(Rayleigh mab) selectively binds to human complement protein C5, inhibiting its cleavage to C5a and C5b during complement activation. This inhibition prevents the release of pro-inflammatory mediators C5a and the formation of the cytolytic pore forming Membrane Attack Complex (MAC) C5b-9, while retaining the proximal or early components of complement activation (e.g., C3 and C3 b) that are critical for opsonization of microorganisms and clearance of immune complexes.

In other embodiments, the antibody comprises

(Rayleigh mab) heavy and light chain CDRs or variable regions. For example, in one embodiment, the antibody comprises a sequence set forth in SEQ ID NO. 12

(Rayleigh mab) CDR1, CDR2 and CDR3 domains of the VH region, and having the sequence shown in SEQ ID NO:8

(Rayleigh mab) CDR1, CDR2 and CDR3 domains of the VL region. In another embodiment, the antibody comprises heavy chain CDR1, CDR2 and CDR3 domains having sequences shown in

SEQ ID NOs

19, 18 and 3, respectively, and light chain CDR1, CDR2 and CDR3 domains having sequences shown in

SEQ ID NOs

4, 5 and 6, respectively. In another embodiment, the antibody comprises VH and VL regions having the amino acid sequences set forth in SEQ ID NO 12 and SEQ ID NO 8, respectively.

Another exemplary anti-C5 antibody is antibody BNJ421, or antigen-binding fragments and variants thereof, comprising a heavy chain and a light chain having the sequences set forth in SEQ ID NOS: 20 and 11, respectively. BNJ421 (also referred to as ALXN 1211) is described in PCT/US2015/019225 and U.S. Pat. No. 9,079,949, the teachings of which are incorporated herein by reference.

In other embodiments, the antibody comprises the heavy and light chain CDRs or variable regions of BNJ 421. Thus, in one embodiment, the antibody comprises the CDR1, CDR2 and CDR3 domains of the BNJ421 VH region having the sequence shown in SEQ ID NO. 12 and the CDR1, CDR2 and CDR3 domains of the BNJ421 VL region having the sequence shown in SEQ ID NO. 8. In another embodiment, the antibody comprises heavy chain CDR1, CDR2 and CDR3 domains having sequences shown in

SEQ ID NOs

The exact boundaries of the CDRs have been defined differently according to different approaches. In some embodiments, the CDRs or frames within the light or heavy chain variable domainsThe positions of the shelf regions may be defined as follows: kabat et al [ (1991) "Sequences of Proteins of Immunological Interest [ protein Sequences of Immunological Interest]"NIH publication No. 91-3242, department of Health and Human Services (U.S., department of Health and Human Services), besserda, md ]. In this case, the CDRs may be referred to as "Kabat CDRs" (e.g., "Kabat LCDR2" or "Kabat HCDR 1"). In some embodiments, the positions of the CDRs of the light or heavy chain variable region may be defined as follows: chothia et al (1989) Nature [ Nature]342:877-883. Thus, these regions may be referred to as "Chothia CDRs" (e.g., "Chothia LCDR2" or "Chothia HCDR 3"). In some embodiments, the positions of the CDRs of the light and heavy chain variable regions can be defined as in the Kabat-Chothia combination definitions. In such embodiments, these regions may be referred to as "combined Kabat-Chothia CDRs". Thomas et al [ (1996) Mol Immunol [ molecular immunology ]]33(17/18):1389-1401]Illustrating the identification of CDR boundaries according to the Kabat and Chothia definitions.

In some embodiments, the anti-C5 antibodies described herein comprise a heavy chain CDR1 comprising or consisting of the amino acid sequence: GHIFSNYWIQ (SEQ ID NO: 19). In some embodiments, the anti-C5 antibodies described herein comprise a heavy chain CDR2 comprising or consisting of the amino acid sequence of seq id no: EILPGSGHTEYTENFKD (SEQ ID NO: 18). In some embodiments, the anti-C5 antibodies described herein comprise a heavy chain variable region comprising the following amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKASGHIFSNYWIQWVRQAPGQGLEWMGEILPGSGHTEYTENFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARYFFGSSPNWYFDVWGQGTLVTVSS (SEQ ID NO: 12).

In some embodiments, the anti-C5 antibodies described herein comprise a light chain variable region comprising the amino acid sequence: DIQMTQSPSSLSASVGDRVTITCGASENIYGALNWYQQKPGKAPKLLIYGATNLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNVLNTPLTFGQGTKVEIK (SEQ ID NO: 8).

Another exemplary anti-C5 antibody is 7086 antibody described in U.S. patent nos. 8,241,628 and 8,883,158. In one embodiment, the antibody comprises the heavy and light chain CDRs or variable regions of the 7086 antibody (see U.S. patent nos. 8,241,628 and 8,883,158). In another embodiment, the antibody or antigen-binding fragment thereof comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences shown in SEQ ID NOs 21, 22, and 23, respectively, and light chain CDR1, CDR2, and CDR3 domains having the sequences shown in SEQ ID NOs 24, 25, and 26, respectively. In another embodiment, the antibody or antigen-binding fragment thereof comprises the VH domain of the 7086 antibody having the sequence shown in SEQ ID NO. 27 and the VL domain of the 7086 antibody having the sequence shown in SEQ ID NO. 28.

Another exemplary anti-C5 antibody is the 8110 antibody also described in U.S. patent nos. 8,241,628 and 8,883,158. In one embodiment, the antibody comprises the heavy and light chain CDRs or variable regions of the 8110 antibody. In another embodiment, the antibody or antigen-binding fragment thereof comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences shown in SEQ ID NOs 29, 30, and 31, respectively, and light chain CDR1, CDR2, and CDR3 domains having the sequences shown in SEQ ID NOs 32, 33, and 34, respectively. In another embodiment, the antibody comprises an 8110 antibody VH domain having the sequence shown in SEQ ID NO. 35 and an 8110 antibody VL domain having the sequence shown in SEQ ID NO. 36.

Another exemplary anti-C5 antibody is the 305LO5 antibody described in US 2016/0176954 A1. In one embodiment, the antibody comprises the heavy and light chain CDRs or variable regions of the 305LO5 antibody. In another embodiment, the antibody or antigen-binding fragment thereof comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences shown in SEQ ID NOs 37, 38, and 39, respectively, and light chain CDR1, CDR2, and CDR3 domains having the sequences shown in SEQ ID NOs 40, 41, and 42, respectively. In another embodiment, the antibody comprises the VH domain of the 305LO5 antibody having the sequence set forth in SEQ ID NO. 43 and the VL domain of the 305LO5 antibody having the sequence set forth in SEQ ID NO. 44.

Another exemplary anti-C5 antibody is Rep [ reported ], 24/4/2017 in Fukuzawa, t.et al; 7 (1): 1080. In one embodiment, the antibody comprises the heavy and light chain CDRs or variable regions of the SKY59 antibody. In another embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising SEQ ID NO 45 and a light chain comprising SEQ ID NO 46.

Another exemplary anti-C5 antibody is the REGN3918 antibody (also known as H4H12166 PP) described in US 20170355757 (see US 10,633,434). In one embodiment, the antibody comprises a heavy chain variable region comprising SEQ ID NO. 47 and a light chain variable region comprising SEQ ID NO. 48. In another embodiment, the antibody comprises a heavy chain comprising SEQ ID NO. 49 and a light chain comprising SEQ ID NO. 50.

In some embodiments, the anti-C5 antibodies described herein may comprise a variant human Fc constant region that binds to a human neonatal Fc receptor (FcRn) with greater affinity for binding to the human neonatal Fc receptor than to a native human Fc constant region from which the variant human Fc constant region is derived. For example, the Fc constant region may comprise one or more (e.g., two, three, four, five, six, seven, or eight or more) amino acid substitutions relative to a native human Fc constant region from which the variant human Fc constant region is derived. Substitutions can increase the binding affinity of IgG antibodies containing the variant Fc constant region for FcRn at pH 6.0 while maintaining the pH dependence of the interaction. Methods for testing whether one or more substitutions in an antibody Fc constant region increase the affinity of the Fc constant region for FcRn at pH 6.0 (while maintaining the pH dependence of the interaction) are known in the art.

Substitutions that enhance the binding affinity of the antibody Fc constant region for FcRn are known in the art and include, for example, the M252Y/S254T/T256E triple substitutions described by (1) Dall' Acqua et al (2006) J Biol Chem [ journal of biochemistry ]281 23514-23524; (2) M428L or T250Q/M428L substitutions described in Hinton et al (2004) J Biol Chem [ journal of biochemistry ] 279; and (3) substitutions N434A or T307/E380A/N434A as described in Petkova et al (2006) Int Immunol [ International immunology ]18 (12): 1759-69. Additional substitution pairings: P257I/Q311I, P I/N434H and D376V/N434H are described, for example, in Datta-Mannan et al (2007) J Biol Chem [ journal of biochemistry ]282 (3): 1709-1717, the disclosure of which is incorporated herein by reference in its entirety.

In some embodiments, the variant constant region has a substitution for valine at EU amino acid residue 255. In some embodiments, the variant constant region has a substitution for asparagine at EU amino acid residue 309. In some embodiments, the variant constant region has a substitution of isoleucine at EU amino acid residue 312. In some embodiments, the variant constant region has a substitution at EU amino acid residue 386.

In some embodiments, the variant Fc constant region comprises no more than 30 (e.g., no more than 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2) amino acid substitutions, insertions, or deletions relative to the native constant region from which it is derived. In some embodiments, the variant Fc constant region comprises one or more amino acid substitutions selected from the group consisting of: M252Y, S254T, T256E, N434S, M428L, V259I, T I and V308F. In some embodiments, the variant human Fc constant region comprises a methionine at position 428 and an asparagine at position 434 of the native human IgG Fc constant region, each numbered in EU. In some embodiments, the variant Fc constant region comprises 428L/434S double substitutions, such as described in U.S. patent No. 8,088,376.

In some embodiments, the precise location of these mutations may be offset from the native human Fc constant region location due to antibody engineering. For example, a 428L/434S double substitution may correspond to that in BNJ441 (when used in an IgG2/4 chimeric Fc)

(rayleigh mab)) and 429L and 435S in the M429L and N435S variants found in U.S. patent No. 9,079,949, the disclosure of which is incorporated by reference herein in its entirety.

In some embodiments, the variant constant region comprises a substitution at amino acid position 237, 238, 239, 248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 325, 332, 334, 360, 376, 380, 382, 384, 385, 386, 387, 389, 424, 428, 433, 434, or 436 (EU numbering) relative to the native human Fc constant region. In some embodiments, the substitution is selected from the group consisting of: a methionine for glycine at position 237; alanine for proline at position 238; a lysine substituted for serine at position 239; isoleucine for lysine at position 248; an alanine, phenylalanine, isoleucine, methionine, glutamine, serine, valine, tryptophan, or tyrosine substituted for threonine at position 250; a phenylalanine, tryptophan, or tyrosine substituted for methionine at position 252; a threonine to serine at position 254; a glutamic acid for arginine at position 255; an aspartic acid, glutamic acid, or glutamine substituted for threonine at position 256; alanine, glycine, isoleucine, leucine, methionine, asparagine, serine, threonine, or valine at position 257 in place of proline; a histidine for glutamic acid at position 258; alanine for aspartic acid at position 265; a phenylalanine in place of aspartic acid at position 270; an alanine or glutamic acid substituted for asparagine at position 286; a histidine for threonine at position 289; alanine for asparagine at position 297; a glycine substituted for serine at position 298; alanine for valine at position 303; alanine for valine at position 305; alanine, aspartic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, valine, tryptophan, or tyrosine for threonine at position 307; alanine, phenylalanine, isoleucine, leucine, methionine, proline, glutamine, or threonine for valine at position 308; alanine, aspartic acid, glutamic acid, proline, or arginine for leucine or valine at position 309; alanine, histidine, or isoleucine for glutamine at position 311; an alanine or histidine for aspartic acid at position 312; a lysine or arginine substituted for leucine at position 314; an alanine or histidine for asparagine at position 315; alanine for lysine at position 317; glycine substituted asparagine at position 325; a valine for isoleucine at position 332; a leucine substituted for lysine at position 334; a histidine for lysine at position 360; an alanine for aspartic acid at position 376; alanine for glutamic acid at position 380; alanine for glutamic acid at position 382; an alanine for asparagine or serine at position 384; an aspartic acid or histidine for glycine at position 385; proline for glutamine at position 386; glutamic acid for proline at position 387; an alanine or serine substituted for asparagine at position 389; alanine for serine at position 424; alanine, aspartic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, asparagine, proline, glutamine, serine, threonine, valine, tryptophan, or tyrosine for methionine at position 428; a lysine in place of histidine at position 433; (iii) an alanine, phenylalanine, histidine, serine, tryptophan, or tyrosine substituted for asparagine at position 434; and histidine for tyrosine or phenylalanine at position 436, all by EU numbering.

In some embodiments, suitable anti-C5 antibodies for use in the methods described herein comprise a heavy chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO. 14 and/or a light chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO. 11. Alternatively, in some embodiments, the anti-C5 antibody used in the methods described herein comprises a heavy chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO. 20 and/or a light chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO. 11.

In one embodiment, the antibody has an affinity dissociation constant (K) of at least 0.1 (e.g., at least 0.15, 0.175, 0.2, 0.25, 0.275, 0.3, 0.325, 0.35, 0.375, 0.4, 0.425, 0.45, 0.475, 0.5, 0.525, 0.55, 0.575, 0.6, 0.625, 0.65, 0.675, 0.7, 0.725, 0.75, 0.775, 0.8, 0.825, 0.85, 0.875, 0.9, 0.925, 0.95, or 0.975) nM at pH 7.4 and 25 ℃ (and, otherwise, under physiological conditions) _D ) Binding to C5. In some embodiments, K of the anti-C5 antibody or antigen binding fragment thereof _D Not greater than 1 (e.g., not greater than 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, or 0.2) nM.

In other embodiments, [ (antibody against K at pH 6.0 and ℃ C ] C5 _D ) /(antibodies against K at pH7.4 and 25 ℃ C5 _D )]Greater than 21 (e.g., greater than 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 8)0. 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, or 8000).

Methods for determining whether an antibody binds to a protein antigen and/or the affinity of an antibody for a protein antigen are known in the art. For example, binding of an antibody to a protein antigen can be detected and/or quantified using a variety of techniques, such as, but not limited to, western blotting, dot blotting, surface Plasmon Resonance (SPR) methods (e.g., BIAcore System; pharmacia biosensor AB, piscataway, sweden and New Jersey) or enzyme-linked immunosorbent assays (ELISA). See, e.g., benny k.c. lo (2004) "Antibody Engineering: methods and Protocols [ Antibody Engineering: methods and protocols ] ", humana Press (ISBN: 1588290921); john et al (1993) J Immunol Meth [ J Immunol methods ] 160; jonsson et al (1993) Ann Biol Clin [ clinical biology Ann ] 51; and Jonsson et al (1991) Biotechniques [ Ann. Biotech. Ann. Rev.11. In addition, methods for measuring affinity (e.g., dissociation and association constants) are set forth in the working examples.

As used herein, the term "k _a "refers to the rate constant of binding of an antibody to an antigen. The term "k _d "refers to the rate constant at which an antibody dissociates from an antibody/antigen complex. And the term "K _D "refers to the equilibrium dissociation constant of an antibody-antigen interaction. The equilibrium dissociation constant is derived from the ratio of kinetic rate constants, K _D ＝k _a /k _d . Such assays are preferably measured at 25 ℃ or 37 ℃ (see working examples). For example, the kinetics of antibody binding to human C5 can be determined by immobilizing the antibody using an anti-Fc capture method by Surface Plasmon Resonance (SPR) on a BIAcore 3000 instrument at pH 8.0, 7.4, 7.0, 6.5 and 6.0.

In one embodiment, the anti-C5 antibody or antigen-binding fragment thereof blocks the production or activity of a C5a and/or C5b active fragment of a C5 protein (e.g., a human C5 protein). Through this blocking effect, the antibody inhibits, for example, the pro-inflammatory effects of C5a and the production of the cell surface C5b-9 Membrane Attack Complex (MAC).

Methods for determining whether a particular antibody or therapeutic agent described herein inhibits C5 cleavage are known in the art. Inhibition of the human complement component C5 reduces the cytolytic capacity of complement in a body fluid of a subject. This reduction in the cytolytic capacity of complement present in the body fluid(s) may be measured by methods well known in the art, e.g., by conventional hemolytic assays, e.g., by Kabat and Mayer (ed), "Experimental Immunochemistry ]"2 nd edition," 135-240, springfield, illinofield, CC Thomas (1961), pp.135-139, or a conventional variant of this assay, e.g.Hillmen et al, (2004) N Engl J Med [ journal of medicine, new England]，350(6)552) by chicken red blood cell hemolysis. Methods for determining whether a candidate compound inhibits cleavage of human C5 into C5a and C5b forms are known in the art and described in Evans et al (1995) Mol Immunol [ molecular immunology]32(16)1183-95. For example, the concentration and/or physiological activity of C5a and C5b in a body fluid can be measured by methods well known in the art. For C5b, one or more hemolytic assays for soluble C5b-9 as discussed herein may be used. Other assays known in the art may also be used. Using these or other suitable types of assays, candidate agents that are capable of inhibiting human complement component C5 can be screened.

Immunological techniques such as, but not limited to, ELISA can be used to measure the protein concentration of C5 and/or its split products to determine the ability of an anti-C5 antibody or antigen-binding fragment thereof to inhibit the conversion of C5 to a biologically active product. In some embodiments, the generation of C5a is measured. In some embodiments, a C5b-9 neo-epitope specific antibody is used to detect the formation of terminal complement.

A hemolytic assay may be used to determine the inhibitory activity of the anti-C5 antibody or antigen binding fragment thereof on complement activation. To determine the effect of anti-C5 antibodies or antigen-binding fragments thereof on classical complement pathway-mediated hemolysis in vitro serum test solutionsFor example, sheep red blood cells coated with hemolysin or chicken red blood cells sensitized with an anti-chicken red blood cell antibody are used as target cells. The percent lysis was normalized by considering 100% lysis to be equal to the lysis that occurred in the absence of inhibitor. In some embodiments, the classical complement pathway is activated by human IgM antibodies, e.g.

Classical pathway complement kit (

COMPL CP310, euro-diagnostic, sweden). Briefly, test sera are incubated with anti-C5 antibodies or antigen-binding fragments thereof in the presence of human IgM antibodies. The amount of C5b-9 produced is measured by contacting the mixture with an enzyme-conjugated anti-C5 b-9 antibody and a fluorogenic substrate and measuring the absorbance at the appropriate wavelength. As a control, test sera were incubated in the absence of anti-C5 antibody or antigen-binding fragment thereof. In some embodiments, the test serum is a serum deficient in C5 reconstituted with a C5 polypeptide.

To determine the effect of anti-C5 antibodies or antigen-binding fragments thereof on alternative pathway-mediated hemolysis, unsensitized rabbit or guinea pig erythrocytes can be used as target cells. In some embodiments, the serum test solution is a serum deficient in C5 reconstituted with a C5 polypeptide. The percent lysis was normalized by considering 100% lysis to be equal to lysis that occurred in the absence of inhibitor. In some embodiments, the alternative complement pathway is activated by a lipopolysaccharide molecule, e.g., as in

Alternative pathway complement kit (

COMPL AP330, euro-diagnostic, sweden). Briefly, test sera are incubated with anti-C5 antibodies or antigen-binding fragments thereof in the presence of lipopolysaccharide. By passingThe amount of C5b-9 produced is measured by contacting the mixture with an enzyme-conjugated anti-C5 b-9 antibody and a fluorogenic substrate and measuring the fluorescence at the appropriate wavelength. As a control, test sera were incubated in the absence of anti-C5 antibody or antigen-binding fragment thereof.

In some embodiments, C5 activity or inhibition thereof is quantified using a CH50eq assay. The CH50eq assay is a method used to measure total classical complement activity in serum. The test is a lysis assay that uses antibody-sensitized erythrocytes as activators of the classical complement pathway and various dilutions of test serum to determine the amount (CH 50) required to provide 50% lysis. For example, the percent hemolysis can be determined using a spectrophotometer. The CH50eq assay provides an indirect measurement of Terminal Complement Complex (TCC) formation, as TCC itself is directly responsible for the measured hemolysis.

Such assays are well known to those skilled in the art and are commonly practiced. Briefly, to activate the classical complement pathway, an undiluted serum sample (e.g., a reconstituted human serum sample) is added to a microassay well containing antibody-sensitized red blood cells, thereby generating TCC. Next, the activated serum is diluted in microassay wells coated with a capture reagent (e.g., an antibody that binds to one or more components of TCC). TCC present in the activated samples bound to monoclonal antibodies coating the surfaces of the microassay wells. Wells were washed and a detection reagent that detectably labeled and recognized bound TCC was added to each well. The detectable label may be, for example, a fluorescent label or an enzymatic label. The results are expressed as CH50 unit equivalents per mL (CH 50U Eq/mL).

For example, inhibition associated with terminal complement activity includes terminal complement activity in a hemolytic assay or CH50eq assay that is reduced by at least 5% (e.g., at least 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60%) as compared to the effect of a control antibody (or antigen-binding fragment thereof) under similar conditions and equimolar concentrations. As used herein, significantly inhibited refers to a given activity (e.g., terminal complement activity) being inhibited by at least 40% (e.g., at least 45%, 50%, 55) %, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or higher). In some embodiments, the anti-C5 antibodies described herein are directed to

Contains one or more amino acid substitutions in the CDR of (i.e., SEQ ID NO: 1-6), but remains in the hemolytic assay or CH50eq assay

At least 30% (e.g., at least 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%) of complement inhibitory activity.

In one embodiment, the antibody competes with the antibodies described herein for binding to the same epitope on C5 and/or for binding to the same epitope on C5. The term "binds to the same epitope" with respect to two or more antibodies means that the antibodies bind to the same fragment of amino acid residues as determined by a given method. Techniques for determining whether an antibody binds to the same epitope on "C5" as an antibody described herein include, for example, epitope mapping, such as for antigens: x-ray analysis of crystals of antibody complexes, these methods provide atomic resolution of epitopes, and hydrogen/deuterium exchange mass spectrometry (HDX-MS). Other methods monitor binding of antibodies to peptide antigen fragments or mutant variants of the antigen, where loss of binding due to modification of amino acid residues within the antigen sequence is generally considered an indicator of epitope composition. Furthermore, computational combinatorial methods for epitope mapping can also be used. These methods rely on the ability of the antibody of interest to affinity isolate a particular short peptide from a combinatorial phage display peptide library. Antibodies with the same VH and VL or the same CDR1, 2 and 3 sequences are expected to bind the same epitope.

An antibody that "competes with another antibody for binding to a target" refers to an antibody that inhibits (partially or completely inhibits) the binding of another antibody to the target. Whether and to what extent two antibodies compete with each other for binding to the target, i.e., whether and to what extent one antibody inhibits the other antibody from binding to the target, can be determined using known competition experiments. In certain embodiments, the antibody competes with another antibody and inhibits binding of the other antibody to the target by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. The level of inhibition or competition may vary depending on which antibody is the "blocking antibody" (i.e., the cold antibody that is first incubated with the objective). The competing antibodies bind to the same epitope, an overlapping epitope, or an adjacent epitope (e.g., as indicated by steric hindrance).

The anti-C5 antibodies or antigen-binding fragments thereof described herein for use in the methods described herein can be generated using a variety of art-recognized techniques. Monoclonal antibodies can be obtained by various techniques familiar to those skilled in the art. Briefly, splenocytes from animals immunized with the desired antigen are generally immortalized by fusion with myeloma cells (see, kohler and Milstein, eur.j. Immunol. [ european journal of immunology ]6, 511-519 (1976)). Alternative immortalization methods include transformation with epstein-barr virus, oncogenes or retroviruses or other methods well known in the art. Colonies from individual immortalized cells are screened to produce antibodies with the desired specificity and affinity for the antigen, and the yield of monoclonal antibodies produced by these cells can be increased by various techniques, including injection into the peritoneal cavity of a vertebrate host. Alternatively, the DNA sequence encoding the monoclonal antibody or binding fragment thereof can be isolated by screening a DNA library from human B cells according to the general protocol outlined in Huse et al, science [ Science ]246 1275-1281 (1989).

Composition IV

The composition can be formulated as a pharmaceutical solution, e.g., for administration to a subject to treat or prevent a complement-associated disorder. The pharmaceutical composition typically includes a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" refers to and includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The compositions may include pharmaceutically acceptable salts, such as acid or base addition salts, sugars, carbohydrates, polyols, and/or tonicity adjusting agents.

The compositions may be formulated according to standard methods. Pharmaceutical formulation is a well-established technique (see, e.g., gennaro (2000) "Remington: the Science and Practice of Pharmacy [ Remington: science and Practice of Pharmacy ]," 20 th edition, lippincott, williams and Wilkisis (ISBN: 0683306472), "Ansel et al (1999)" Pharmaceutical Dosaage Forms and Drug Delivery Systems [ Drug Dosage Forms and Drug Delivery Systems ], "7 th edition, lippincott Williams and Wilkins publisher (ISBN: 0683305727)," and "Kibbe (2000)" Handbook of Pharmaceutical Excipients American Pharmaceutical Association of Pharmaceutical Association [ Drug adjuvants ], "3 rd edition (ISBN: 091733096X 36) ]. In some embodiments, the composition can be, for example, formulated as a buffer solution at a suitable concentration and suitable for storage at 2 ℃ -8 ℃ (e.g., 4 ℃). In some embodiments, the composition can be formulated for storage at temperatures below 0 ℃ (e.g., -20 ℃ or-80 ℃). In some embodiments, the composition can be formulated for storage at 2 ℃ -8 ℃ (e.g., 4 ℃) for up to 2 years (e.g., 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 11/2 years, or 2 years). Thus, in some embodiments, the compositions described herein are stable for at least 1 year when stored at 2 ℃ -8 ℃ (e.g., 4 ℃).

The pharmaceutical composition may be in various forms. These forms include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories. The preferred form will depend in part on the intended mode of administration and therapeutic application. For example, compositions containing compositions for systemic or local delivery may be in the form of injectable or infusible solutions. Thus, the compositions can be formulated for parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular injection). As used herein, "parenteral administration," "parenteral administration," and other grammatically equivalent phrases refer to modes of administration other than enteral and topical administration, typically by injection, and includes, but is not limited to, intravenous, intranasal, intraocular, pulmonary, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intrapulmonary, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intracerebral, intracranial, carotid, and intrasternal injection and infusion.

V. Process

Provided herein are methods for treating PNH in a subject who has previously exhibited an inadequate response to anti-C5 antibody therapy by administering to the subject a therapeutically effective amount of an inhibitor of AP. In some embodiments, the inhibitor of AP is an inhibitor that inhibits a target upstream of C5, such as factor D or complement 3 (C3).

In some embodiments, the treatment decreases one or more of: (a) persistent extravascular hemolysis (EVH); (b) anemia; and/or (c) transfusion-dependent; and/or improvement in FACIT fatigue scale score. In some embodiments, control of MAC-mediated intravascular hemolysis is maintained or improved in insufficiently responsive PNH subjects after treatment.

In some embodiments, an inadequate response to anti-C5 antibody therapy is associated with: pharmacokinetic (PK) aspects, e.g., (a) ineffective inhibition of C5 cleavage in a subject; (b) Low dose of anti-C5 antibody and/or low subject plasma levels; (C) enhanced clearance of anti-C5 antibodies in the subject; and/or (d) the anti-C5 antibody intolerance in the subject results in a reduction in anti-C5 antibody dose, preferably wherein the anti-C5 antibody intolerance comprises fatigue and post-infusion pain. In some embodiments, an inadequate response to anti-C5 antibody therapy is associated with: pharmacodynamic (PD) aspects, e.g., (a) CR1 polymorphism; (b) Extravascular hemolysis (EVH), e.g., opsonization of surviving blood cells by intravascular hemolysis (IVH); and/or (C) the deleterious effect of the C3 fragment on the activity of the anti-C5 antibody. In some embodiments, an inadequate response to anti-C5 antibody therapy is associated with one or more PK and PD aspects.

Also provided herein are methods for treating PNH in a human patient, comprising administering to the patient a CFD inhibitor alone or in combination with an anti-C5 antibody or antigen-binding fragment thereof. In some embodiments, the CFD inhibitor and/or the anti-C5 antibody or antigen-binding fragment thereof is administered (or is for administration) according to a particular clinical dosage regimen (e.g., at a particular dose and according to a particular dosing regimen).

In one embodiment, a method is provided for treating PNH in a subject, the method comprising: administering to the subject a therapeutically effective amount of a Complement Factor D (CFD) inhibitor in combination with a therapeutically effective amount of an anti-C5 antibody or antigen-binding fragment thereof,

(b) Transfusion independence; and/or

wherein the inadequate response of the subject is transfusion dependence and/or anemia; and is

(d) An increase in hemoglobin of 2.0g/dL or more compared to a baseline hemoglobin level of the subject;

(e) Transfusion independence or avoidance of transfusion; and/or

(f) The FACIT fatigue scale score is increased by 10 points or more compared to the subject's baseline FACIT fatigue scale score.

wherein the inadequate response of the subject is transfusion dependence and/or anemia; and is provided with

(b) Transfusion independence or avoidance of transfusion; and/or

In some embodiments, the methods further comprise determining the hemoglobin level, transfusion status, and/or FACIT fatigue scale score of the subject at baseline and at 12 and/or 24 weeks post-treatment, wherein (a) hemoglobin is increased by 2.0g/dL or more compared to the subject's baseline hemoglobin level; (b) transfusion independent or avoidance of transfusion; and/or (c) an increase in the FACIT fatigue scale score of 10 points or more compared to the subject's baseline FACIT fatigue scale score is indicative of treatment. In some embodiments, the methods involve treating a subject with PNH who has previously been treated with an anti-C5 antibody therapy (e.g.,

An insufficient response was shown. In some embodiments, a subject with PNH has previously been on an approved dose or higher without changing the regimen ≦ 8 weeks

Exhibiting an inadequate response for more than or equal to 24 weeks (e.g., 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more weeks). In some embodiments, the subject with PNH was previously paired with

An insufficient response was shown.

In some embodiments, the inadequate response of the subject is transfusion-dependent (e.g., greater than or equal to 1 Red Blood Cell (RBC) transfusion performed at a time ≦ 12 weeks prior to screening). In some embodiments, the inadequate response of the subject is anemia (e.g., hemoglobin <10 g/dl). In some embodiments, the inadequate response of the subject is transfusion dependence and anemia.

In some embodiments, the method further comprises determining the subject's hemoglobin level, transfusion status, and/or FACIT fatigue scale score at baseline and at 12 and/or 24 weeks post-treatment, wherein

(b) Transfusion independence or avoidance of transfusion; and/or

(c) An increase in the FACIT fatigue scale score of 10 points or more compared to the subject's baseline FACIT fatigue scale score is indicative of treatment.

In some embodiments, a CFD inhibitor (e.g., danicopan) is administered (or is used to administer) according to a particular clinical dosage regimen (e.g., at a particular dose and according to a particular dosing regimen). In some embodiments, the CFD inhibitor is administered to the subject orally. In some embodiments, the CFD inhibitor is orally administered to the subject three times daily (TID). In some embodiments, the CFD inhibitor is administered to the subject orally at a dose of about 50mg to 300 mg. In some embodiments, the CFD inhibitor is administered orally at a dose of about 100mg, 110mg, 120mg, 130mg, 140mg, 150mg, 160mg, 170mg, 180mg, 190mg, 200mg, 210mg, 220mg, 230mg, 240mg, 250mg, 260mg, 270mg, 280mg, 290mg, or 300 mg. In some embodiments, the CFD inhibitor is administered orally at a dose of about 100 mg. In some embodiments, the CFD inhibitor is administered orally at a dose TID of about 100 mg. In some embodiments, the CFD inhibitor is administered orally at a dose of about 150 mg. In some embodiments, the CFD inhibitor is administered orally at a dose TID of about 150 mg. In some embodiments, the CFD inhibitor is administered orally at a dose of about 200 mg. In some embodiments, the CFD inhibitor is administered orally at a dose TID of about 200 mg.

In some embodiments, the anti-C5 antibody or antigen-binding fragment thereof is administered (or is used to administer) according to a particular clinical dosage regimen (e.g., at a particular dose and according to a particular dosing regimen) (e.g.,

or

In some embodiments, the anti-C5 antibody or antigen-binding fragment thereof is administered to the subject at a dose of 600mg per week, four times, followed by 900mg at week 5, and thereafter 900mg every 2 weeks. In some embodiments, the method will comprise

In some embodiments, the anti-C5 antibody or antigen-binding fragment thereof is administered to a subject younger than 18 years of age and weighing 40kg and above at a dose of 900mg per week, four times, then 1200mg at week 5, and 1200mg every two weeks thereafter. In some embodiments, will

In some embodiments, the anti-C5 antibody or antigen-binding fragment thereof is administered to a subject less than 18 years of age and weighing 30kg to less than 40kg at a dose of 600mg per week, twice per week, followed by 900mg at week 3 and 900mg every two weeks thereafter. In some embodiments, will

In some embodiments, the anti-C5 antibody or antigen-binding fragment thereof is administered at a dose of 600mg per weekIn subjects less than 18 years old and weighing from 20kg to less than 30kg, twice, then at week 3 at a dose of 600mg, and thereafter at a dose of 600mg every two weeks. In some embodiments, the method will comprise

Subjects less than 18 years old and weighing 20kg to less than 30kg are administered twice a week at a dose of 600mg, then at a dose of 600mg on week 3, and thereafter at a dose of 600mg every two weeks.

Subjects less than 18 years old and weighing 5kg to less than 10kg were administered once a week at a dose of 300mg, then at a dose of 300mg on week 2, and thereafter at a dose of 300mg every three weeks.

In some embodiments, the dose of the anti-C5 antibody or antigen-binding fragment thereof is based on the weight of the patient. In some embodiments, for example, 300mg of the anti-C5 antibody or antigen-binding fragment thereof is administered to a patient weighing ≧ 5 to <10 kg. In some embodiments, 600mg of the anti-C5 antibody or antigen-binding fragment thereof is administered to a patient weighing ≧ 10 to <20 kg. In some embodiments, 900mg or 2100mg of the anti-C5 antibody or antigen-binding fragment thereof is administered to a patient weighing ≧ 20 to <30 kg. In some embodiments, 1200mg or 2700mg of the anti-C5 antibody or antigen-binding fragment thereof is administered to a patient that weighs ≧ 30 to <40 kg. In some embodiments, 2400mg or 3000mg of an anti-C5 antibody or antigen-binding fragment thereof is administered to a patient weighing ≧ 40 to <60 kg. In some embodiments, 2700mg or 3300mg of the anti-C5 antibody, or antigen-binding fragment thereof, is administered to a patient weighing ≧ 60 to <100 kg. In some embodiments, 3000mg or 3600mg of the anti-C5 antibody or antigen-binding fragment thereof is administered to a patient weighing ≧ 100 kg. In certain embodiments, the dosage regimen is adjusted to provide the best desired response (e.g., an effective response).

In some embodiments of the present invention, the,

the following applications were performed:

(a) Once on day 1 of the administration cycle, at a dose of 2400mg; and

In some embodiments, the method will comprise

Applied to the body weight of more than or equal to 40<60kg of patients:

(a) Once on day 1 of the administration cycle, at a dose of 2400mg; and

(a) Once on day 1 of the administration cycle, the dose was 2700mg; and

In some embodiments, will

Applied to the body weight of more than or equal to 60 to<100kg of patients:

(a) Once on day 1 of the administration cycle, the dose was 2700mg; and

(a) Once on day 1 of the administration cycle, at a dose of 3000mg; and

In some embodiments, will

Applied to patients with the weight more than or equal to 100 kg:

(a) Once on day 1 of the administration cycle, at a dose of 3000mg; and

(a) Once on day 1, at the following doses: 600mg of a patient with a weight of more than or equal to 5 to less than 10kg, 600mg of a patient with a weight of more than or equal to 10 to less than 20kg, 900mg of a patient with a weight of more than or equal to 20 to less than 30kg, 1200mg of a patient with a weight of more than or equal to 30 to less than 40kg, 2400mg of a patient with a weight of more than or equal to 40 to less than 60kg, 2700mg of a patient with a weight of more than or equal to 60 to less than 100kg, or 3000mg of a patient with a weight of more than or equal to 100 kg; and

(b) On day 15 and every four weeks thereafter, the doses were: 300mg of patients with the body weight of more than or equal to 5 to less than 10kg, or 600mg of patients with the body weight of more than or equal to 10 to less than 20 kg; or on day 15 and every eight weeks thereafter, the dose is: 2100mg of patients with a weight of more than or equal to 20 to less than 30kg, 2700mg of patients with a weight of more than or equal to 30 to less than 40kg, 3000mg of patients with a weight of more than or equal to 40 to less than 60kg, 3300mg of patients with a weight of more than or equal to 60 to less than 100kg, or 3600mg of patients with a weight of more than or equal to 100 kg.

In some embodiments, the anti-C5 antibody or antigen-binding fragment thereof is administered to a body weight ≧ 5 to<10kg of patients: once on day 1 at a dose of 600mg; and (b) at day 15 and every four weeks thereafter, at a dose of 300mg. In some embodiments, the method will comprise

In some embodiments, the anti-C5 antibody or antigen-binding fragment thereof is administered to a body weight ≧ 20 to<30kg of patients: once on day 1, at a dose of 900mg; and (b) a dose of 2100mg on day 15 and every eight weeks thereafter. In some embodiments, the method will comprise

In some embodiments, the anti-C5 antibody or antigen-binding fragment thereof is administered to a body weight ≧ 30<40kg of patients: once on day 1 at a dose of 1200mg; and (b) on day 15 and every eight weeks thereafter, at a dose of 2700mg. In some embodiments, the method will comprise

In some embodiments, the anti-C5 antibody or antigen-binding fragment thereof is administered to a body weight ≧ 40 to<60kg of patients: once on day 1, dose was 2400mg; and (b) at day 15 and every eight weeks thereafter, at a dose of 3000mg. In some embodiments, the method will comprise

In some embodiments, the anti-C5 antibody or antigen-binding fragment thereof is administered to a body weight ≧ 60 to<100kg of patients: once on day 1, the dose was 2700mg; and (b) on day 15 and every eight weeks thereafter, at a dose of 3300mg. In some embodiments, will

In some embodiments, to obtain an effective response, the anti-C5 antibody or antigen-binding fragment thereof is administered to the patient in an amount and at a frequency to maintain at least 50 μ g, 55 μ g, 60 μ g, 65 μ g, 70 μ g, 75 μ g, 80 μ g, 85 μ g, 90 μ g, 95 μ g, 100 μ g, 105 μ g, 110 μ g, 115 μ g, 120 μ g, 125 μ g, 130 μ g, 135 μ g, 140 μ g, 145 μ g, 150 μ g, 155 μ g, 160 μ g, 165 μ g, 170 μ g,175 μ g, 180 μ g, 185 μ g, 190 μ g, 195 μ g, 200 μ g, 205 μ g, 210 μ g, 215 μ g, 220 μ g, 225 μ g, 230 μ g, 235 μ g, 240 μ g, 245 μ g, 250 μ g, 255 μ g, or 260 μ g of antibody per milliliter of patient's blood. In some embodiments, the anti-C5 antibody is administered to the patient in an amount and at a frequency that maintains between 50 μ g and 250 μ g antibody per milliliter of patient blood. In some embodiments, the anti-C5 antibody is administered to the patient in an amount and at a frequency to maintain 100 μ g to 200 μ g antibody per milliliter of patient blood. In some embodiments, the anti-C5 antibody is administered to the patient in an amount and at a frequency that maintains about 175 μ g of antibody per milliliter of patient blood.

In one embodiment, a method for use in the past is provided

administering to a subject a therapeutically effective amount of a danicopan in combination with a therapeutically effective amount of

(eculizumab) was prepared by mixing the following components,

wherein will be

(eculizumab) is administered intravenously to the subject at a dose of 600mg weekly, four times, followed by 900mg at week 5, and thereafter 900mg every 2 weeks; and is

(b) Transfusion independence or avoidance of transfusion; and/or

(eculizumab) was added to the reaction mixture,

wherein will be

(b) Administering to a subject weighing 30kg to less than 40kg twice a week at a dose of 600mg, followed by a dose of 900mg at week 3, and thereafter at a dose of 900mg every two weeks;

(e) Administering to a subject weighing 5kg to less than 10kg once a week at a dose of 300mg, followed by a dose of 300mg at week 2, and thereafter at a dose of 300mg every three weeks; and is

An increase in hemoglobin of 2.0g/dL or higher compared to a baseline hemoglobin level of the subject;

transfusion independent or avoidance; and/or

In another embodiment, a method for treating PNH in a subject is provided, the method comprising:

(eculizumab) was added to the reaction mixture,

wherein will be

(eculizumab) is administered to the subject intravenously at a dose of 600mg weekly, four times, followed by a dose of 900mg at week 5, and thereafter 900mg every 2 weeks; and is

Wherein the subject exhibits one or more of the following clinical improvements 12 and/or 24 weeks after treatment with the CFD inhibitor;

(b) Transfusion independence or avoidance of transfusion; and/or

(eculizumab) was added to the reaction mixture,

wherein will be

(eculizumab) intravenous administration:

(e) Administering to a subject weighing 5kg to less than 10kg once a week at a dose of 300mg, followed by a dose of 300mg on week 2, and thereafter at a dose of 300mg every three weeks; and is

transfusion independent or avoidance; and/or

In some embodiments, the methods described herein further comprise determining the hemoglobin level, transfusion status, and/or FACIT fatigue scale score of the subject at baseline and at 12 and/or 24 weeks post-treatment, wherein (a) there is an increase in hemoglobin of 2.0g/dL or more as compared to the subject's baseline hemoglobin level; (b) transfusion independent or avoidance of transfusion; and/or (c) an increase in the FACIT fatigue scale score of 10 points or more compared to the subject's baseline FACIT fatigue scale score is indicative of treatment.

Results VI

Provided herein are methods for treating PNH in a patient. Symptoms of PNH include, but are not limited to, pallor, fatigue (e.g., tiredness, difficulty with daily activity, difficulty concentrating, dizziness, weakness), pain (e.g., stomach pain, leg pain or swelling, chest pain, back pain), dark urine, shortness of breath, difficulty swallowing, yellowing of skin and/or eyes, anemia, cytopenia, erectile dysfunction, blood clots, kidney disease, organ damage, stroke, or heart attack.

Patients treated according to the methods disclosed herein experience an improvement in at least one sign of PNH. The treatment can produce at least one therapeutic effect selected from the group consisting of pallor, fatigue, jaundice, anemia, cytopenia, abdominal pain, dyspnea, dysphagia, chest pain, or reduction or cessation of erectile dysfunction.

In some embodiments, the subject exhibits one or more additional clinical improvements after treatment according to the methods described herein. For example, in one embodiment, after treatment, the subject exhibits an increase in hemoglobin of 2.0g/dL or greater compared to the subject's baseline hemoglobin level. In some embodiments, after 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 weeks of treatment, the subject exhibits an increase in hemoglobin of 2.0g/dL or greater compared to the subject's baseline hemoglobin level. In some embodiments, the subject exhibits an increase in hemoglobin of 2.0g/dL or more compared to the subject's baseline hemoglobin level after 24 weeks of treatment.

In some embodiments, after treatment, the subject exhibits transfusion independence. In some embodiments, the subject exhibits transfusion independence after 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 weeks of treatment. In some embodiments, the subject exhibits transfusion independence after 24 weeks of treatment. In some embodiments, the subject exhibits avoidance of blood transfusion after treatment. In some embodiments, the subject exhibits avoidance of blood transfusion after 12 weeks of treatment.

In some embodiments, after treatment, the subject exhibits a 10 point or more increase in the FACIT fatigue scale score (e.g., 10, 11, 12) compared to the subject's baseline FACIT fatigue scale score. In some embodiments, the subject exhibits a 10 point or more increase in the FACIT fatigue scale score after 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 weeks of treatment. In some embodiments, the subject exhibits a 10 point or more increase in the FACIT fatigue scale score after 12 and/or 24 weeks of treatment.

In some embodiments, after treatment (e.g., after 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 weeks), the subject exhibits an increase in hemoglobin of 2.0g/dL as compared to the subject's baseline hemoglobin level and exhibits transfusion independence. In some embodiments, the subject exhibits a 2.0g/dL increase in hemoglobin compared to the subject's baseline hemoglobin level after 12 and/or 24 weeks of treatment and exhibits transfusion independence.

In some embodiments, the subject exhibits an increase in hemoglobin of 2.0g/dL and an increase in the FACIT fatigue scale score of 10 points or more after treatment (e.g., after 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 weeks of treatment) as compared to the subject's baseline hemoglobin level. In some embodiments, the subject exhibits an increase in hemoglobin of 2.0g/dL compared to the subject's baseline hemoglobin level after 12 and/or 24 weeks of treatment and exhibits a 10 point or more increase in the FACIT fatigue scale score.

In some embodiments, after treatment (e.g., after 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 weeks of treatment), the subject exhibits transfusion independence or avoidance of transfusions and an increase in the FACIT fatigue scale score of 10 points or more. In some embodiments, the subject exhibits transfusion independence or avoidance of transfusions and a 10 point or more increase in FACIT fatigue scale score after 12 or 24 weeks of treatment

In some embodiments, after treatment (e.g., after 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 weeks of treatment), the subject exhibits an increase in hemoglobin of 2.0g/dL compared to the subject's baseline hemoglobin level, and exhibits transfusion independence or avoidance of transfusions and an increase in the FACIT fatigue scale score of 10 points or more. In some embodiments, the subject exhibits an increase in hemoglobin of 2.0g/dL after 12 or 24 weeks of treatment, and exhibits a 10 point or more increase in transfusion independence or avoidance and FACIT fatigue scale score, as compared to the subject's baseline hemoglobin level.

In some embodiments, treatment shifts bilirubin to normal levels (e.g., from about 0.2-1.2 mg/dL).

In some embodiments, the treatment results in a reduction (e.g., a 2, 3, 4, or 5 fold reduction) of reticulocytes compared to baseline.

In some embodiments, the treatment results in an increase (e.g., 2, 3, 4, or 5 fold increase) in PNH-specific erythrocyte clone size compared to baseline.

In some embodiments, treatment results in a reduction (e.g., 2, 3, 4, or 5 fold reduction) of PNH red blood cells opsonized with the C3 fragment as compared to baseline.

In some embodiments, the treatment results in a reduced need for transfusion as compared to baseline.

In some embodiments, the treatment is such that blood transfusion is avoided.

In some embodiments, the treatment results in terminal complement inhibition.

In some embodiments, the treatment shifts at least one or more hemolysis-associated blood biomarker selected from the group consisting of: free hemoglobin, haptoglobin, reticulocyte count, PNH Red Blood Cell (RBC) clone, and/or D-dimer.

In some embodiments, lactate Dehydrogenase (LDH) levels can be used to assess responsiveness to therapy (e.g., a decrease in hemolysis assessed by Lactate Dehydrogenase (LDH) levels indicates an improvement in at least one sign of PNH). LDH is a marker of intravascular hemolysis (Hill, a. Et al, br.j. Haematol. [ journal of hematology in uk ], 149. Red Blood cells contain large amounts of LDH and correlations between cell-free hemoglobin and LDH concentrations have been reported in vitro (Van Lente, f. et al, clin. Chem. [ clinical chemistry ], 27. The consequences of hemolysis are not associated with anemia (Hill, A. Et al, haematologica [ hematology ],93 (s 1): 359Abs.0903,2008, kanakura, Y. Et al, int.J. Hematol. [ J. Hematology of International hematology ], 93. LDH concentrations obtained continuously at baseline and subsequently throughout the treatment period are important measures of hemolysis. Baseline levels of cell-free plasma hemoglobin were significantly elevated in PNH patients with LDH greater than or equal to 1.5 times the upper limit of normal (LDH greater than or equal to 1.5 × ULN), and there was a significant correlation between LDH and cell-free plasma hemoglobin (Hillmen, p. Et al, n.engl.j.med. [ new england journal of medicine ], 355. Normal LDH values ranged from 105-333IU/L (International units/liter).

LDH levels can be measured using any suitable test or assay, for example, ferri's Clinical Advisor edited by Ferri FF [ Ferri Clinical guidelines ]2014.Philadelphia pa; 2014, section IV-Laboratory tests and interpretation of results. LDH concentrations can be measured in various samples obtained from patients, in particular in serum samples. As used herein, the term "sample" refers to a biological material from a subject. Although serum LDH concentrations are of concern, the sample may be from other sources, including, for example, a single cell, multiple cells, a tissue, a tumor, a biological fluid, a biomolecule, or a supernatant or extract of any of the foregoing. Examples include tissue removed for biopsy, tissue removed during resection, blood, urine, lymphoid tissue, lymph fluid, cerebrospinal fluid, mucus, and stool samples. The sample used may vary depending on the format of the assay, the method of detection, and the nature of the tumor, tissue, cell, or extract to be assayed. Methods for preparing samples are known in the art and can be readily adjusted to obtain samples compatible with the method used.

In some embodiments, LDH levels in patients treated according to the disclosed methods are reduced to near normal levels or to within 10%, 20%, 30%, 40%, or 50% lower than the values considered normal levels (e.g., within the range of 105-333IU/L (international units/liter)). In some embodiments, the LDH level of the patient is normalized throughout the treatment maintenance period. In some embodiments, the LDH level of the treated patient is normalized for at least 95% of the time during the maintenance of treatment. In some embodiments, the LDH level of the treated patient is normalized for at least 90%, 85%, or 80% of the time during treatment maintenance. In some embodiments, prior to initiation of treatment, the patient's LDH level is greater than or equal to 1.5 times the upper normal limit (LDH greater than or equal to 1.5 × ULN).

In some embodiments, treatment results in a reduction of major adverse vascular events (MAVE; e.g., thrombophlebitis/deep vein thrombosis, pulmonary embolism, myocardial infarction, transient ischemic attack, unstable angina, renal vein thrombosis/renal artery thrombosis/glomerular thrombosis, renal infarction, acute peripheral vascular occlusion, mesenteric/visceral vein/arterial thrombosis or infarction, hepatic/portal vein thrombosis, cerebral artery occlusion/cerebrovascular accident, cerebral vein occlusion, renal artery thrombosis, or multi-infarct dementia).

In some embodiments, the treatment shifts to normal levels a biomarker associated with the chronic disease, the biomarker selected from the group consisting of: estimated glomerular filtration rate (eGFR) and field urine Albumin creatinine and plasma Brain Natriuretic Peptide (BNP).

In some embodiments, the Treatment alters the Quality of Life relative to baseline as assessed by the Quality of Life 30 Core Questionnaire Scale (Quality of Life questonaire-Core 30 Scale) of the 4 th edition and European Cancer Research and Treatment organization (European organization for Research and Treatment of Cancer).

VII. Kit

Also provided herein are kits for treating PNH. The kit optionally may further comprise instructions, for example, comprising an administration schedule to allow a practitioner (e.g., a physician, nurse, or patient) to administer the composition(s) contained therein, thereby administering the composition(s) to a patient having PNH. The kit may also include a syringe.

Optionally, the kit comprises multiple packages of single dose pharmaceutical composition(s), each package comprising an effective amount of a CFD inhibitor and/or an anti-C5 antibody for administration according to the methods provided above. The instruments or equipment required for administering the pharmaceutical composition(s) may also be included in the kit. For example, the kit may provide one or more pre-filled syringes containing an effective amount of a CFD inhibitor and/or an anti-C5 antibody.

In some embodiments, the kit comprises: (a) A dose of a Complement Factor D (CFD) inhibitor and (b) instructions for using CFD in any of the methods described herein. In some embodiments, the kit comprises: (a) a dose of a Complement Factor D (CFD) inhibitor; (b) dosage of anti-C5 antibody; and (C) instructions for using the CFD and anti-C5 antibody in any of these methods described herein. In some embodiments, the CFD is danicopan. In some embodiments, the anti-C5 antibody is eculizumab (e.g.,

) Or Rayleigh mab

The following examples are illustrative only, and should not be construed as limiting the scope of the disclosure in any way, as many variations and equivalents will become apparent to those skilled in the art upon reading the disclosure. The contents of all references, genbank entries, patents, and published patent applications cited throughout this application are expressly incorporated by reference herein.

Examples of the invention

Example 1: danikopan (ACH 4471) in the same pair

Phase 2 open label study in patients with Paroxysmal Nocturnal Hemoglobinuria (PNH) with inadequate response to monotherapy

During the period from 6 months to 9 months in 2018 to 2019, the pairs

Phase 2 clinical studies were performed on PNH patients who did not respond adequately to monotherapy (eculizumab). In particular, the aim of the study was to evaluate patients with PNH (in pairs) who had been administered the inhibitor danicopan, a factor D inhibitor

(eculizumab) suboptimal response) blood transfusion requirements.

A.Method of producing a composite material

1. Patient's health

Except that it continues with its current view shown in fig. 1

Out of the (eculizumab) regimen, 12 were up to 65 years old and on 200mg TID with dose escalation in response

(Ekulizumab) exhibited a suboptimal response (i.e., hemoglobin [ Hgb)]<10g/dL and transfusion dependence [ screening ≤ 12 weeks for ≥ 1 RBC infusion]) Is administered orally three times daily (TID) 100-150mg of danicopan (ACH-4471; ACH-0144471). Key inclusion and exclusion criteria are shown in table 2.

Table 2: key inclusion and exclusion criteria

Specifically, the patient orally took danicopan three times daily at a starting dose of 100mg or 150mg and was instructed to take these doses about the same time daily, and as close to 8 hours apart as possible. All doses were taken about 15-30 minutes after meal or snack consumption. Based on safety and hemoglobin values, the dose was escalated to a maximum of 200mg three times daily and allowed to increase by 50mg every 4 weeks during cycle 12. If the patient has not reached a maximum dose of 200mg three times daily by week 12, escalation is allowed if clinically indicated. Patients continued to use their preexisting ones throughout the study

(eculizumab) protocol. During the 24 week treatment period, the patient is not allowed to use

(eculizumab) to another C5 inhibitor. Patients who completed treatment with clinical benefit entered the long-term extension phase.

2. Endpoint

The primary objective of this study was to evaluate danicopan and its dose based on the increase in Hgb at week 24 of Treatment (TW) relative to baseline

(eculizumab) standard of care efficacy.Secondary goals included RBC unit reduction during 24 weeks of daunorubin treatment compared to 24 weeks prior to daunorubin treatment, and RBC infusion independent patient percentage during 24 weeks of daunorubin treatment, as well as changes in Lactate Dehydrogenase (LDH) at TW24 relative to baseline. Additional endpoints included the effect of danicopan on complement biomarkers and fatigue assessment evaluated using a FACIT fatigue tester with a total score ranging from 0 to 52 points, with higher scores indicating improvement (see, e.g., cella D et al, cancer [ Cancer ] evaluation]2002;94 (2):528-538). The general safety, tolerability, pharmacokinetics and pharmacodynamics of danekan were determined. After 24 weeks of complete treatment, the patient entered a long-term extension period.

3. Pharmacokinetic and pharmacodynamic methods

Plasma danisco Pan Nongdu was determined by protein precipitation: to a 75 μ L plasma aliquot (K2 EDTA as anticoagulant) was added an acetonitrile solution containing 0.15% formic acid, followed by liquid chromatography/tandem mass spectrometry in positive ionization mode using a deuterated internal standard for quantification. Ion transitions of 580.2 to 360.2amu and 587.2 to 362.2amu of danicopan and internal standard were monitored, respectively.

Pharmacodynamics was determined by measuring serum AP activity by AP hemolysis assay. Serum CP activity, plasma Bb concentrations, serum FD, and C3 concentrations were also monitored. Complement testing was performed by a central laboratory using commercial kits, except for the AP hemolysis assay, which was performed internally for exploration purposes. In each AP hemolysis run, a single normal human serum sample is included in addition to the patient serum sample, and thus the hemolysis value of a single patient serum sample can be normalized to that of a normal human serum sample. Finally, PNH clone size and C3 fragment deposition on erythrocytes were measured using flow cytometry with FITC conjugated anti-human C3d antibody.

4. Statistical method

Descriptive statistical data is provided for biochemical, quality of life measurements and transfusion data. The continuous variables are summarized with mean, median, minimum and maximum values. Categorical variables, such as transfusion independent variables, were summarized in counts and percentages. Missing values were not estimated.

Transfusion frequency and blood volume were assessed via annual rate and units, respectively. The average intensity rate of pre-treatment transfusion frequency was calculated by adding the number of transfusion events within 52 weeks prior to screening to the number of transfusions of 10 patients from screening to day 1 of administration, and dividing this sum by the total exposure time of the patients. The mean intensity rate at 24 weeks after treatment was calculated in the same manner. The ratio of these two intensity rates (posterior to anterior) was used to quantify the effect of treatment. Standard statistical analysis was performed to compare the two intensity rates (post-treatment versus pre-treatment). The reduction in blood volume was assessed by annual transfusion units using the same procedure.

B.Results

1. Patient characteristics

12 patients were enrolled in the study and received at least one dose of danekang. One patient was discontinued after taking 2 doses of darunavir due to a severe adverse event that is unlikely to be associated with potential exacerbations (pulmonary hypertension/edema) of the darunavir. The patient had a prior presence of pulmonary hypertension (valvular hypertension and due to their PNH) and their cardiac drugs had changed the first few days before the study drug was started. Severe adverse events were considered unlikely to be related to study drug and patient data were excluded from the analysis.

11 patients completed treatment. The treatment regimen is shown in table 3. The results are shown in Table 4. The median age was 42.5 years and the mean C5 treatment duration before dosing was 36 months. All patients adopt stable

(eculizumab) protocol, wherein 8 patients were injected intravenously every 14 days with an approved dose of 900mg. Two patients take 1200mg

(eculizumab), and one patient took 1500mg every 14 days.

Despite the use of stabilising

(eculizumab) therapy, but with a slight increase in LDH levels at the time of patient admission. Patients were anemic, with a mean Hgb of 7.9mg/dL (SD ± 1.5 g/dL), and all but one patient had a history of RBC transfusions, with a mean of 3.4 transfusions (mean 5.8 units) over the 24 week period prior to screening. Patients without history of transfusion did not receive transfusion due to religious dissatisfaction; she began with a baseline Hgb of 5.0g/dL (patient A, table 4) and was diagnosed with hereditary elliptocytosis.

Table 3: treatment regimens

Table 4: key clinical parameters at baseline, week 12 and week 24

F-female; hgb-hemoglobin; IV-intravenous; LDH-lactate dehydrogenase; m-male; PNH-paroxysmal nocturnal hemoglobinuria; q14 d-every 14 days; RBC-red blood cells; TID-three times daily.

* Score based on chronic disease treatment Function Assessment (FACIT) -fatigue scale V4. The fraction ranges from 0 to 52. Scores less than 30 indicate severe fatigue.

The subject was not tested for C3 fragment deposition at week 12. No data is input.

Sample stability disruption

^§ N =7; for four patients, the samples were out of stability.

^$ Geometric mean (range)

2. Research disposal

Nine patients initially took 100mg of danilazone every 8 hours, and two patients initially took 150mg of danilazone every 8 hours. Of the patients who initially took 100mg, one patient titrated 200mg every 8 hours, and six patients titrated 150mg every 8 hours, with two patients titrating 150mg titrating 200mg every 8 hours at the end of the trial. All patients maintained it throughout the study

(Ekulizumab) protocol, but one patient in the United states improved on Hgb, before week 16, his insurance company took him

(eculizumab) dose was reduced (from 1200mg to 900 mg). One patient was discontinued after taking 2 doses of danimod due to the above-mentioned severe adverse events unlikely to be associated with danimod, and the remaining 11 patients reached the primary endpoint at the time of treatment and were included in the final analysis.

3. Pharmacokinetics

C of Daniscopan at steady state during day of intensive pharmacokinetic sampling on 11 patients on week 7 _max 、T _max And AUC _(0-8hr) The average values (% CV) of (A) were 432 (37) ng/mL, 2.14 (33) hr and 1806 (37) ng · hr/mL, respectively. During these dense sampling days,% CV C was averaged _Grain The value was 105 (57) ng/mL. Sampling was performed for the remainder of the study duration under less controlled conditions, resulting in an average (% CV) C of 150 (59) ng/mL _Grain The value is obtained. These results are consistent with the pharmacokinetic values of danicopan observed in healthy volunteer studies.

4. Clinical curative effect

As shown in table 4, benefits were observed in multiple laboratory markers of PNH. Hgb increased by an average of 2.4g/dL at 24 weeks of treatment. In most patients, this therapeutic effect on hemoglobin appeared at week 2 and was maintained during the study.

In addition to the observed elevation of hemoglobin in patients receiving dannikon, a clinically significant reduction in RBC transfusion requirements was also demonstrated during the 24-week treatment period, as shown in figure 2. Of ten patients with a history of 52 weeks transfusions, 57 transfusions were performed on a total of 101 units of Packed Red Blood Cells (PRBC) by the time of screening and between the screening and dosing days (day 1), based on historical data. Note that one patient (patient a) was excluded from the analysis because the patient refused transfusion due to religious objection. Since the start of the treatment with danicopan, only one patient received a single transfusion at

week

24, 2 units total, administered during hospitalization for pneumonia, which researchers believe was associated with danicopan Pan Moguan. The mean of the annual rates of transfusion events were 5.234 (before administration of danipran) and 0.217 (after administration of danipran), the ratio (after administration of danipran and before administration of danipran) was 0.042 (95% CI =0,0.176 p = 0.0001), indicating a statistically very significant 95.8% reduction in transfusion frequency after administration of danipran (see fig. 3). The mean values for the aged transfusion units were 9.27 (before administration of danicopan) and 0.43 (after administration of danicopan), the ratio (after administration of danicopan and before administration of danicopan) was 0.047 (95% CI =0,0.224 p = 0.0019), which also reflects a very significant reduction in the number of transfusion units due to the addition of danicopan (see fig. 3).

In addition to improvements in hemoglobin and transfusion requirements, improvements in clinically relevant parameters were observed (table 4). In the study, patients receiving danicopan treatment continued to have a further improvement in LDH than the upper normal limit, and also had a significant improvement in absolute reticulocyte count. A decrease in total bilirubin and direct bilirubin measurements in patients with elevated baseline levels was also observed in the study.

The study used the validated quality of life tool FACIT fatigue Scale (4 th edition). The self-reporting tool measures the degree of fatigue experienced on a scale of 0 to 52, with scores less than 30 indicating severe fatigue and higher scores indicating improved fatigue. A 3 point change is clinically significant on this scale. A 10 point or higher variation is very significant on this scale. Reporting a FACIT fatigue score, with

The mean increase at 24 weeks was 11 points (table 4) compared to baseline (eculizumab). The greatest improvement was observed in patient F (a 44 year old female subject) who improved 43 points from baseline to week 24 (i.e., 9 points at baseline versus 52 points at week 24). This constitutes an upper limit of the present disclosure.

Complement biomarkers were monitored during the course of the study. Serum FD and C3 concentrations were normal at baseline and there was little change during danilaplon treatment (data not shown). Inhibition of CP activity was nearly complete at baseline, indicating the presence of a small amount of free serum C5 (fig. 4A). This was present throughout the study. In contrast, residual AP activity was detected at baseline with the AP hemolysis assay and decreased after administration of danicopan (fig. 4B). At the same time, administration of dannikoe Pan Houxie also decreased the level of plasma Bb (fig. 4C).

In agreement with previous reports by Hill et al, evaluation was made

(Ekuzhu)Mab), the percentage of PNH RBCs opsonized with C3 fragments at baseline (i.e.,% C3 d) due to accumulation of C3 fragments on PNH RBCs that survive intravascular hemolysis in the presence of a C5 inhibitor ⁺ PNH RBC) higher (geometric mean 22%; in the range 6.3-80%) (see, e.g., hill et al, haematologica [ hematology ]]2010; 95:567-573). Addition of danisco Pan Xianzhu reduced the percentage of PNH RBCs conditioned with C3 fragments (geometric mean 6.7%; range 0.1% -57% at week 24) (fig. 4D). Meanwhile, the clone size of PNH RBC increased from 54 + -24.9% at baseline to 84 + -22.1% at 24 weeks (mean + -SD), approaching that of PNH granulocytes, which was higher at baseline (mean + -SD: 92 + -9.2%), and remained higher during the study (mean + -SD: 95 + -7.2% at 24 weeks) (FIG. 4D).

5. Safety feature

The tolerability of dannikonpan is generally good. At least 2 patients reported adverse event therapy emergencies (TEAEs) are listed in table 5, and 96% of the treated TEAEs were mild to moderate in severity. There was no interruption by TEAE.

The severity of all events was considered mild to moderate, except for the following 2 patients. One patient presented with grade 3 direct bilirubin elevation on day 70, concurrent with grade 1 ALT elevation. The patient's elevation in these parameters was similar during baseline and screening. By day 77, both adverse events were resolved. The dosage of danicopan was temporarily decreased and then re-escalated after the event resolved. The patient completed the study. Researchers believe that these events are caused by breakthrough hemolysis because the associated LDH approximately doubles and Hgb decreases by 0.8mg/dL.

The second patient experienced a severe adverse pneumonia event on day 145, required hospitalization, and recovered on day 152. This event has evolved from viral bronchitis. The patient also had a history of neutropenia. The patient received 2 units of PRBC transfusion during hospitalization in an institution independent of the test center. The relationship to study drug is considered unlikely. Danisco Pan Jiliang was unchanged and the patient completed the study.

Table 5: safety feature

MedDRA preferred terminology reported by more than 10% of patients	n(％)
		Number of patients reporting TEAE	11(100)
Headache (headache)	3(27)
		Abdominal pain	2(18)
Contusion injury	2(18)
		Cough with a symptom of the lung	2(18)
Fatigue	2(18)
		Nasopharyngitis	2(18)
Nausea	2(18)
		Oropharynx	2(18)
Pain in the extremities	2(18)

C.Summary of the invention

Use of

(Ekulizumab) or

C5 inhibition (current standard of care) of (rayleigh mab) is an effective treatment for PNH patients. While this treatment controls intravascular hemolysis and significantly improves overall survival, many patients remain anemic, and some patients may continue to rely on blood transfusion due to persistent extravascular hemolysis.

In the above clinical trials, the background of these severely anemic patients, mainly due to extravascular hemolysis (EVH)

Addition of dacenidae Pan Shi to therapy (eculizumab) increased Hgb by an average of 2.4 grams and reduced RBC transfusion requirements clinically and statistically significantly. When the patient is at

The mean increase in FACIT fatigue score of 11 points was evident and very significant when this experiment was performed in the context of (Ekulizumab) therapy. The international PNH registry shows that fatigue is one of the most common patient-reported symptoms in untreated patients, with about 80% of patients reporting fatigue over the last six months (see, e.g., schrezenmeier h]2014;99 (5): 922-929). For anemic patients, such as those with cancer and PNH patients, fatigue is usually assessed using the FACIT fatigue scale. By using

(Ekulizumab) monotherapy treatment improved the level of fatigue in PNH patients as measured by FACIT fatigue as shown by the marker tests SHEPHERD and TRIUMPH, with a significant increase in scores of 12.2 and 6.4 points, respectively, over baseline (see, e.g., brodsky RA et al, blood]2008;111 (4) 1840-1847 and Hillmen P et al, N.Engl.J.Med. [ New England journal of medicine]2006; 355:1233-1243). In this experiment, among patients with persistent anemia, the patient with persistent anemia was

The addition of dannipagin (Ekulizumab) not only raised hemoglobin by more than 2g/dL, but also indicated that the potential effect of the addition of dannipagin on the life quality of patients.

In this study, the

The addition of dactinopam (eculizumab) almost eliminated the transfusion requirement for most patients. One patient included in the trial had no history of blood transfusions due to religious objections. At the time of the test, she pairs

The baseline hemoglobin of (Ekulizumab) was 5g/dL. At her place

(eculizumab) the addition of dannipagin to her therapy raised her hemoglobin by more than 3g/dL at 24 weeks and significantly improved her fatigue. She was also diagnosed with hereditary oval polycythemia, another hemolytic anemia, but on which daniscan had no effect.

Bilirubin and reticulocytes were also significantly improved. Total bilirubin entered the normal range at week 24 compared to baseline, while reticulocyte count decreased to near normal at week 24. Additionally, PNH erythrocyte clone size was close to that of PNH granulocytes, indicating that the addition of danicok Pan Jin one step protected PNH RBC from hemolysis. The percentage of PNH RBCs opsonized with C3 fragments decreases from baseline to week 24, supporting the upstream AP mechanism of action of danicopan. All these suggest that the use of dannikopan prevents C3-mediated extravascular hemolysis while maintaining control of MAC-mediated intravascular hemolysis.

In summary, except for

In addition to the standard of care for (eculizumab), proof of concept was established with danecopan during the treatment of PNH. Danicopan is generally well tolerated and has significant improvements in Hgb, transfusion demand, FACIT fatigue and other relevant parameters. This indicates that it is receiving

Further benefit can be obtained in standard of care patients (eculizumab) by using AP of danisco Pan Zuduan factor D. This benefit may be due to prevention of C3-mediated extravascular hemolysis, while controlling intravascular hemolysis. Danicopan addresses an unmet need for PNH.

Example 2: phase 3 study of clinically significant extravascular hemolysis (EVH) of Danicopan in patients with paroxysmal nocturnal hemoglobinuria as an adjunct therapy to C5 inhibitors

A phase 3 clinical study was conducted on adult PNH patients (over 18 years old) with clinically significant extravascular hemolysis (EVH), substantially in accordance with the protocol, the entire disclosure of which is incorporated herein by reference.

A.Target

The primary objective of this study was to evaluate danicopan (also known as "ALXN2040" and ACH 4471) as a C5 inhibitor (i.e., as a C5 inhibitor) at 12 weeks

Or

) Compared with oral (tablet) placebo. The primary endpoint was the change in hemoglobin (Hgb) from baseline after 12 weeks of treatment with dannikon compared to placebo.

Key secondary goals include assessing the proportion of patients who avoid transfusion, the change in chronic disease treatment Function Assessment (FACIT) fatigue score from baseline, and the change in absolute reticulocyte count from baseline.

Evaluation: (1) As an additional therapy to C5 inhibitors compared to placebo, danekan had an impact on avoiding transfusions at week 12 (i.e., the proportion of patients who avoid Transfusions (TA) is defined as patients who remain transfused within week 12 and do not need to be transfused according to the guidelines prescribed by the protocol); (2) Effect of danicopan on FACIT fatigue score at 12 weeks of treatment (i.e., change in FACIT fatigue score at 12 weeks from baseline) as an additional therapy to C5 inhibitor compared to placebo; and (3) effect of danecopan on absolute reticulocyte count (i.e., change in absolute reticulocyte count at week 12 from baseline) as an adjunct therapy to C5 inhibitors compared to placebo.

Additional goals include the assessment: (1) As an adjunct therapy to C5 inhibitors, danico Pan Duijie is affected by transfusion requirements at 24 weeks in those patients treated with danicopan for 24 weeks

(i.e., the number of Red Blood Cell (RBC) units transfused and the variation in transfusion instances between 24 cycles of treatment with danilamp, and the percentage of patients who avoided transfusions after 24 weeks of treatment, compared to 24 weeks before the start of treatment with danilamp); (2) Effect of danilamp on transfusion need at 12 weeks compared to placebo as an additional therapy to C5 inhibitor (i.e. change in the number of RBC units transfused and transfusion instances within 12 weeks of treatment with danilamp compared to 12 weeks of treatment with placebo); (3) Effect of danicopan on FACIT fatigue score after 24 weeks of treatment (i.e., change in FACIT fatigue score at week 24 in all patients from baseline) as an additional therapy to C5 inhibitors. Further objectives include the evaluation of: (1) As an adjunct therapy to C5 inhibitors, the effect of danilamp on hemoglobin stabilization (i.e., the percentage of hemoglobin stabilized patients over the past 12 weeks of treatment in patients receiving danilamp for 24 weeks) and (2) other laboratory markers associated with PNH patients (i.e., the change in total bilirubin and direct bilirubin from baseline in patients treated with danilamp at 12 weeks, the change in PNH RBC clone size, the deposition of C3 fragments on PNH RBCs, and the measurement of alternative pathway activity at 12 weeks of treatment with danilamp, the change in Lactate Dehydrogenase (LDH) and classical pathway activity at 12 weeks, and the percentage of patients normalized for hemoglobin at 12 weeks and 24 weeks, compared to placebo).

Exploratory goals include assessing Patient Reported Outcomes (PRO) and other health-related Quality of Life (QoL) measurements (i.e., change in third-stage EuroQoL5 dimension (EQ-5D-3L) score from baseline at week 12 compared to placebo, change in EQ-5D-3L score from baseline at week 24, change in European Cancer Research and Treatment organization (European organization for Research and Treatment of Cancer (EORTC)) Quality of Life 30 Core Questionnaire Scale (Quality of Life questance-Core 30Scale (QLQ-C30)) from baseline at week 12 compared to placebo), change in EORTC-QLQ-C30 Scale from baseline at week 24 compared to placebo: change in work efficiency and activity Questionnaire from baseline at week 24: change in WPU versus baseline at week 12: change in WPP from baseline at week 12: change in WPU (SHP) from baseline 12, change in HRP at week 12: change in WPP from baseline at week 24: change in WPP from baseline at week 12: change in WPP.

The security objectives include the evaluation: (1) As an additional therapy for C5 inhibitors, safety and tolerability of treatment with danilan for 24 weeks (i.e., the incidence of Treatment Emergent Adverse Events (TEAE), severe Adverse Events (SAE), laboratory abnormalities, and events leading to study drug withdrawal during the 12-week blind and subsequent 12-week open label treatment period), and (2) safety and tolerability of danilan as an additional therapy for C5 inhibitors during LTE (i.e., the incidence of Treatment Emergent Adverse Events (TEAE), severe Adverse Events (SAE), laboratory abnormalities, and events leading to study drug withdrawal).

B.Overall design

This multi-regional, randomized, double-blind, placebo-controlled, multi-dose, phase 3 study was directed to PNH patients with clinically significant EVH using a C5 inhibitor (eculizumab or rayleigh mab). This study included approximately 84 patients who were receiving C5 inhibitor therapy according to a conventional dose and schedule. These patients were enrolled in the study and treated with danicopan or placebo (ratio 2:1).

Randomized cohorts were stratified by transfusion history (i.e., >2 or ≦ 2 transfusions within 6 months post-screening) and Hgb (i.e., <8.5g/dL and ≧ 8.5 g/dL) and japanese patients (defined as patients enrolled into the study from japan)/non-japanese patients. In addition to its C5 inhibitor therapy (eculizumab or rayleigh mab), patients were randomized to three times daily administration of danicopan (tid) or three times daily administration of placebo at a ratio of 2:1 for 12 weeks (treatment period 1). At week 12, patients randomized to placebo were again switched to da nikoke Pan Zhiliao for an additional 12 weeks (treatment period 2), and patients randomized to da nikopan treatment continued to receive 12 additional weeks of da nikopan treatment while continuing with C5 inhibitor therapy. At the end of the treatment period (week 24), the patient may enter a Long Term Extension (LTE) period and continue to receive danicopamil C5 inhibitor therapy.

In this study, patients had received C5 inhibitor therapy for a period of time sufficient to benefit substantially from the therapy, but were still anemic. Long-term therapy with C5 inhibitors alone is not expected to have an additional effect on their clinical response. Historical transfusion requirements and pre-transfusion hemoglobin levels were captured 52 weeks prior to screening visits. These historical data were used to assess the efficacy and safety of the combination therapy in this study.

Screening visits were made no earlier than 4 weeks after transfusion to minimize the impact of transfusion on screening Hgb levels for stratification purposes. Patients were evaluated for vaccination history. All patients were vaccinated against meningococcal infection within 3 years prior to or at the time of initiation of study drug. Patients who begin study drug treatment less than 2 weeks after receiving the meningococcal vaccine must receive appropriate prophylactic antibiotic treatment until 2 weeks after vaccination.

The starting dose of dannikon or placebo was 150mg three times daily. Any patient with alanine aminotransferase or direct bilirubin screening values >1.5 × Upper Limit of Normal (ULN) was dosed with 100mg three times daily. Patients with gilbert syndrome certification documents begin treatment three times daily at the recommended starting dose of 150 mg. Each dose level requires at least 4 weeks of treatment before any subsequent escalation to the next dose level. Based on safety and clinical efficacy, doses can be escalated in 50mg increments up to 200mg three times daily at the time points specified in the protocol (

weeks

4, 8 and 12). All dose escalations obtained after the visit at week 12 were performed on a patient basis. The maximum dose for treatment period 2 was 200mg three times daily. Patients were unable to switch from the C5 inhibitor on day 1 to any other C5 inhibitor during the first 24 weeks of the study, but could do so during LTE.

During the study, the C5 inhibitor + placebo group was escalated in the same manner as C5 inhibitor + danac Pan Zu to maintain blindness. After week 12, the C5 inhibitor + placebo group switched placebo to receiving danicopan treatment during treatment period 2.

All patients returned to the clinic for safety and other assessments during treatment and during LTE as shown in tables 6-8 of example 3. After treatment period 2 (24 weeks) is over, patients can enter LTE for up to 1 year, at the same dose as they received at week 24 in danco Pan Jiliang, plus their C5 inhibitor therapy. During LTE, the patient's dose may be escalated up to 200mg three times daily.

If the patient stops the study, the dosage of danicopan or placebo is gradually reduced over 6 days (1 st and 2 nd reduced visits) and two follow-up visits are made about 14 and 28 days after the last study medication. Patients continued to receive their C5 inhibitor therapy at the same dose and interval as during the decrement visit and follow-up visit.

Patients were randomized to receive danicopan or placebo treatment in addition to their C5 inhibitor therapy at a rate of 2:1 for 12 weeks (treatment period 1). At week 12, patients randomized to placebo treatment switched to receiving danicok Pan Zhiliao for an additional 12 weeks (treatment period 2), and patients randomized to danicopan treatment continued to receive 12 additional weeks of treatment. At the end of the treatment period (24 weeks), the patient may enter the LTE phase, at the same dose plus their C5 inhibitor therapy. Any patient who stops the study at any time point should receive 6 days of reduced treatment, and an additional 28 days of follow-up.

The C5 inhibitor (eculizumab or rayleigh mab) used in this study was considered as background therapy. New drugs used in this way are also considered background therapy if the patient switches from eculizumab to rayleigh mab 24 weeks after completion of treatment, as the patient needs to take a stable dose and interval of their C5 inhibitor for a long time before the study starts.

Inclusion studies required the following criteria:

diagnosis of PNH

2. Clinically significant extravascular haemolysis (EVH) is defined as:

anemia (Hgb. Ltoreq.9.5 g/dL), absolute reticulocyte count ≥ 120X 10 ⁹ /L。

RBC enrichment or Whole blood infusion of at least 1 time within 6 months before study initiation

3. C5 inhibitor treatment was received at the approved dose (or higher) for at least 6 months prior to study day 1, and there was no change in dose or interval during the 224 weeks prior to study day 1. For those patients who have recently converted from eculizumab to rayleigh mab, they must receive at least a loading dose and 3 maintenance doses (at least 24 weeks) of rayleigh mab prior to day 1.

4. The platelet count was 30,000/. Mu.L or more, and platelet infusion was not required.

5. The absolute neutrophil count is greater than or equal to 750/μ L.

6. Document on certification of vaccination with neisseria meningitidis: all patients had to be vaccinated against meningococcal infection within 3 years prior to or at the time of the initial study drug. Patients who begin study drug therapy less than 2 weeks after receiving a meningococcal vaccine must receive appropriate prophylactic antibiotic treatment until 2 weeks after vaccination.

7. Age 18 years or older (or greater than or equal to the lowest adult age according to local legal requirements)

8. Female patients with fertility potential must agree to use a highly effective or acceptable method of contraception within 30 days from the date the informed consent was signed until the last time they took the study medication. Female patients with fertility potential must also be tested for negative serum pregnancy during screening and negative urine pregnancy on day 1.

9. Female patients with documented non-fertility potential do not require contraceptive methods.

10. Male non-sterile patients must agree to use an effective or acceptable method of contraception with the partner(s) having fertility potential from the first day of administration to 90 days after their last study medication. The surgically infertile men need not employ additional contraception. The men must agree not to donate sperm within 90 days of inclusion in the study and their last study drug administration.

11. Informed consent can be signed, including compliance with the informed consent and the requirements and limitations set forth in the present protocol.

12. Emergency medical care must be obtained.

Patients were excluded from the study based on the following criteria:

1. history of major organ transplantation (e.g., heart, lung, kidney, liver) or history of Hematopoietic Stem Cell Transplantation (HSCT).

2. Patients with known aplastic anemia or other bone marrow failure, in need of HSCT or other treatment, including anti-thymocyte globulin and/or immunosuppressive agents

3. Another study agent other than the C5 inhibitor (eculizumab or rayleigh mab) was received within 30 days prior to the start of the study or within 5 half-lives of the study agent (whichever is greater).

1. Known or suspected complement deficiency.

2. Active bacterial or viral infections occurred within 14 days prior to the first study drug administration, with body temperatures >38 ℃ measured for two consecutive days, with other signs of infection, or with any history of fever.

3. The history or presence of any clinically relevant co-morbidities may render a patient unsuitable for study (e.g., may lead to deterioration of the patient's condition, affect the patient's safety during the study, or confound the study results).

4. Laboratory abnormalities at screening, including:

·ALP>2×ULN

·ALT>2×ULN

direct bilirubin >2 × ULN (unless the investigator believes it is due to extravascular hemolysis) and gilbert syndrome patients may be admitted to the study; however, there is a need for a document of gilbert syndrome. If bilirubin is elevated, which is indicative of Gilbert syndrome, the patient is unable to provide documentation; the patient is tested for this condition (see below for details).

8. Any other clinically significant laboratory abnormality judged by the researcher in the opinion of the primary researcher would render the patient unsuitable for the study or at undue risk.

9. A female who is pregnant, lactating, or is scheduled to become pregnant during the study period or within 90 days of study drug administration.

10. Existing evidence of cholestasis.

11. There is evidence of human immunodeficiency virus, hepatitis B, or active hepatitis C infection at the time of screening.

12. Estimated glomerular filtration Rate (eGFR)<30mL/min/1.73m ² And/or is undergoing dialysis.

13. Hypersensitive to the test drug (danecopan) or any of its excipients.

Example 3: a multi-regional, randomized, double-blind, placebo-controlled, multi-dose, phase 3 study was initiated on PNH patients with clinically significant EVH using a C5 inhibitor (eculizumab or rayleigh mab). This study included approximately 84 patients who were receiving C5 inhibitor therapy according to a conventional dose and schedule. Randomized cohorts were stratified by transfusion history (e.g., >2 or ≦ 2 transfusions within 6 months post-screening) and Hgb (e.g., <8.5g/dL and ≧ 8.5 g/dL) and japanese patients (defined as patients enrolled into the study from japan)/non-japanese patients.

In addition to its C5 inhibitor therapy (eculizumab or rayleigh mab), patients were randomized to either administration of dannikopan (tid) or administration of placebo (tid) at a ratio of 2:1 for 12 weeks (treatment period 1). At week 12, patients randomized to placebo treatment switched to receiving danicok Pan Zhiliao for an additional 12 weeks (treatment period 2), and patients randomized to danicopan treatment will continue to receive danicopan treatment for an additional 12 weeks while continuing with C5 inhibitor therapy. At the end of the treatment period (week 24), the patient may enter a Long Term Extension (LTE) period and continue to receive dannikon + his C5 inhibitor therapy.

In this study, PNH patients had received C5 inhibitor therapy for a period of time sufficient to benefit fully from therapy, but were still anemic. Long-term therapy with C5 inhibitors alone is not expected to have an additional effect on their clinical response. Historical transfusion requirements and pre-transfusion hemoglobin levels can be captured 52 weeks prior to the screening visit. These historical data can be used to assess the efficacy and safety of the combination therapy in this study.

Screening visits were made no earlier than 4 weeks after transfusion to minimize the effect of transfusion on screening Hgb levels to be used for stratification purposes.

Patients will be evaluated for vaccination history. All patients had to be vaccinated against meningococcal infection within 3 years prior to or at the time of the initial study drug. Patients who begin study drug treatment less than 2 weeks after receiving the meningococcal vaccine must receive appropriate prophylactic antibiotic treatment until 2 weeks after vaccination.

The starting dose of danipon or placebo was 150mg tid. Any patient with alanine aminotransferase or direct bilirubin screening values >1.5 × upper normal limit (ULN) will begin treatment at a dosing dose of 100mg tid. Patients with gilbert syndrome certification documents begin treatment at the recommended starting dose of 150mg tid. Each dose level will require at least 4 weeks of treatment before any subsequent escalation to the next dose level. Based on safety and clinical efficacy, the dose can be escalated in 50mg increments up to 200mg tid at the time points specified by the protocol (

weeks

4, 8 and 12). All dose escalations obtained after visit at week 12 were performed by the primary investigator on a patient basis following consultation with the trial committee. The maximum dose for treatment period 2 was 200mg tid. Patients may not switch from their day 1C 5 inhibitor to any other C5 inhibitor in the first 24 weeks of the study, but may do so during LTE.

All patients will return to the clinic for safety and other assessments during treatment and during LTE as shown in tables 6-8.

Evaluating timetables

Table 6: evaluation schedule treatment period 1: all patients were treated

AE = adverse event; AP = alternative pathway; APH = AP hemolysis; CP = classical pathway; ECG = electrocardiogram; FD = factor D; FSH = follicle stimulating hormone; hbs Ag = hepatitis b surface antigen; HCV = hepatitis c virus; HIV Ab = human immunodeficiency virus antibody; HRU = medical resource utilization; INR = international normalized rate; PK = pharmacokinetics; PT = prothrombin time; PTT = partial thromboplastin time; qoL = quality of life; RBC = red blood cells; SAE = severe adverse event.

¹ Visit window was ± 1 day from week 1 to 12. The patient can stop the study at any time and should complete 24 weeks at the last visitAll evaluations

²

Weeks

4, 8 and 12 are potential dose escalation time points.

³ If dose escalation occurs, alanine aminotransferase, aspartate aminotransferase, gamma-glutamyl transferase and alkaline phosphatase should be measured from the hospital facility or from an outpatient blood draw 72 to 96 hours after the escalation.

⁴ Patients will be electrically charged on site within 1 to 3 days, confirmed for hospitalization evaluations, and evaluated for AE, SAE and concomitant medications.

⁵ FSH in postmenopausal women.

⁶ Prior to administration of any vaccine or enhancer, female patients with fertility potential who received the vaccine or enhancer must undergo a negative urine pregnancy test on the day of vaccination.

⁷ The patient will have sufficient study medication available to continue until their next visit. At the time of a dose escalation visit, the patient will return to the clinic to be assigned study medication and receive new instructions for administration.

⁸ Screening and general physical examination on day 1. Simple physical examinations were performed at all other time points.

⁹ The patient will be monitored for fever at each clinic visit and will be self-monitored between visits.

¹⁰ Patients must fast for 8 hours prior to screening, day 1 and blood draw for all visit medical assessments.

¹¹ The patient should avoid vigorous exercise 24 hours prior to blood collection. Walking and light exercise are acceptable.

¹² Serum pregnancy tests were performed at screening. Urine pregnancy tests were performed only on women with fertility potential. On day 1, the urine pregnancy test had to be negative before administration to continue. Any positive urine pregnancy test will be confirmed by a follow-up serum pregnancy test.

¹³ According to the time of clinic visit, the patient will be gotObtaining a pre-administration or post-administration sample; the actual sampling time prior to sample collection and the most recent time of administration should be recorded. The samples should be collected at the same time as the collection of FD, C3 and CP active samples ¹⁴ Refer to the PRO and QoL questionnaires. HRU will be administered on day 1 and week 12

Table 7: evaluation schedule treatment period 2: all patients were

AE = adverse event; AP = alternative pathway; APH = AP hemolysis; CP = classical pathway; ECG = electrocardiogram; EORTC-QLQ = European Cancer Research and Treatment organization (European organization for Research and Treatment of Cancer) Quality of Life 30 item Core Questionnaire Scale (Quality of Life questingnaire-Core 30 Scale); ET = early termination; FD = factor D; HRU = medical resource utilization; INR = international normalized rate; PK = pharmacokinetics; PT = prothrombin time; PTT = partial thromboplastin time;

QoL = quality of life; RBC = red blood cells; SAE = severe adverse events; SHP = work efficiency and activity disorder questionnaire: specific health issues.

¹ Visit window was ± 1 day from week 13 to 24.

² Alanine aminotransferase, aspartate aminotransferase, gamma-glutamyltransferase and alkaline phosphatase should be measured 72 to 96 hours after the dose escalation, if any, by the healthcare facility or by outpatient blood draws.

³ Patients will be electrically charged on site within 1 to 3 days, confirmed for hospitalization evaluations, and evaluated for AE, SAE and concomitant medications.

⁴ Prior to administration of any vaccine or enhancer, female patients with fertility potential who received the vaccine or enhancer must undergo a negative urine pregnancy test on the day of vaccination.

⁵ The patient will have sufficient study medication available to continueUntil the next visit. At the time of a dose escalation visit, the patient will return to the clinic to be assigned study medication and receive new instructions for administration.

⁶ The patient will be monitored for fever at each clinic visit and will be self-monitored between visits.

⁷ Patients must fast for 8 hours on week 24 and before blood draw for all visit medical assessments.

⁸ The patient should avoid vigorous exercise 24 hours prior to blood collection. Walking and light exercise are acceptable.

⁹ Urine pregnancy tests were performed only on women with fertility potential.

¹⁰ Depending on the time of the clinic visit, pre-or post-dose samples will be obtained on site. The actual sampling time before sample collection and the most recent time of administration should be recorded. Samples should be taken at the same time as the collection of FD, C3, CP activity samples.

¹¹ Reference PRO and QoL questionnaires — medical resource utilization (HRU), EORTC-QLQ, and WPAI SHP collect only at week 24

Table 8: evaluation schedule: long term extension, decrement and follow-up periods

AE = adverse event; AP = alternative pathway; APH = AP hemolysis; CP = classical pathway; FD = factor D; F/U = follow-up; INR = international normalized ratio; PT = prothrombin time; PTT = partial thromboplastin time; qoL = quality of life; RBC = red blood cells; SAE = severe adverse events; t = decrement; VHA = visit medical assessment.

¹ The visit window was ± 7 days. Patients must fast for 8 hours before blood sampling.Patients will be electrically challenged on site within 1 to 3 days, samples are confirmed and AE, SAE and concomitant medications are evaluated.

² The visit window was ± 7 days.

⁴ Any patient who stops study medication will complete the decrement and follow-up periods. If the patient exits the study for any reason, all prematurely terminated patients should follow the week 24/ET visit assessment.

⁵ The patient will have sufficient study medication available to continue until their next visit. At the time of a dose escalation visit, the patient will return to the clinic to be dispensed with study medication and receive new instructions for administration.

⁷ PRO and QoL questionnaires will be issued at weeks 40, 56 and 72.

⁹ Any positive test will be confirmed by a follow-up serum pregnancy test.

¹⁰ The test will be performed at weeks 40, 56 and 72

¹¹ Pre-or post-dose samples will be obtained on-site, depending on the time of the clinic visit. The actual sampling time before sample collection and the most recent time of administration should be recorded. Samples should be taken at the same time the FD, C3, CP activity samples are taken.

¹² F/U1: physical examination, vital sign assessment, all necessary safety laboratory tests, and blood and urine sample collection will be performed 2 weeks after the 2 nd decrement period is complete

¹³ F/U2: last one isPhysical examination, assessment of vital signs, all necessary safety laboratory tests and collection of blood and urine samples will be performed 4 weeks after each study medication

After treatment period 2 (week 24) is over, the patient can enter LTE for up to 1 year, at the same dose as that received at week 24 in danco Pan Jiliang, plus their C5 inhibitor therapy. During LTE, the patient may be escalated in dose, up to 200mg tid, at the discretion of the primary researcher and negotiated with the trial principal.

If the patient stops the study, the dosage of the danicopan or placebo should be gradually reduced over 6 days (1 st and 2 nd reduced visits) and two follow-up visits will be made about 14 and 28 days after the last study drug administration. Patients will continue to receive their C5 inhibitor therapy at the same dose and interval as during the decrement visit and follow-up visit.

Intervention group and duration: patients were randomized to receive either dannikon treatment or placebo treatment at a rate of 2:1 for 12 weeks in addition to their C5 inhibitor therapy (treatment period 1). At week 12, patients randomized to placebo treatment will switch to receiving danico Pan Zhiliao for an additional 12 weeks (treatment period 2), and patients randomized to danicopan treatment will continue to receive treatment for an additional 12 weeks. At the end of the treatment period (24 weeks), patients may enter LTE phase, the same dose plus their C5 inhibitor therapy. Any patient who stopped the study at any time point received 6 days of reduced treatment, and an additional 28 days of follow-up.

The C5 inhibitor (eculizumab or rayleigh mab) used in this study will be considered as background therapy.

Inclusion criteria were:

diagnosis of PNH

2. Clinically significant extravascular haemolysis (EVH) is defined as:

3. C5 inhibitor treatment was received at the approved dose (or higher) for at least 6 months prior to study day 1, and there was no change in dose or interval during the 224 weeks prior to study day 1. For those patients who have recently converted from eculizumab to rayleigh mab, they must receive at least a loading dose and 3 maintenance doses (at least 24 weeks) of rayleigh mab before day 1.

4. The platelet count is greater than or equal to 30,000/μ L, and platelet infusion is not required.

5. The absolute neutrophil count is greater than or equal to 750/μ L.

6. Document for verification of neisseria meningitidis vaccination: all patients had to be vaccinated against meningococcal infection within 3 years prior to or at the time of the initial study drug. Patients who begin study drug treatment less than 2 weeks after receiving the meningococcal vaccine must receive appropriate prophylactic antibiotic treatment until 2 weeks after vaccination.

10. Non-sterile male patients must agree to use an effective or acceptable method of contraception with the partner(s) having fertility potential from the first day of administration to 90 days after their last study medication.

Surgically infertile men do not need to take additional contraception.

The men must agree not to donate sperm for 90 days after inclusion in the study and their last study medication.

12. Emergency medical care must be obtained.

Exclusion criteria:

3. Another study agent other than the C5 inhibitor (eculizumab or rayleigh mab) was received within 30 days before the start of the study or within 5 half-lives of the study agent (whichever is larger).

4. Known or suspected complement deficiency.

5. Active bacterial or viral infections occurred within 14 days prior to the first study drug administration, with body temperatures >38 ℃ measured for two consecutive days, with other signs of infection, or with any history of fever.

6. The history or presence of any clinically relevant co-morbidity may render the patient unsuitable for the study (e.g., may lead to deterioration of the patient's condition, affect the patient's safety during the study, or confound the study results).

7. Laboratory abnormalities at screening, including:

alkaline phosphatase >2 × Upper Normal Limit (ULN)

Alanine aminotransferase >2 × ULN

Direct bilirubin >2 × ULN (unless the investigator believes that a patient will be admitted to the study due to extravascular hemolysis and Gilbert syndrome; however, a proof of Gilbert syndrome is required. If elevated bilirubin suggests Gilbert syndrome, but the patient cannot provide a proof, then the patient is tested for this condition.

10. Existing evidence of cholestasis.

11. There is evidence of human immunodeficiency virus, hepatitis B, or active hepatitis C infection in the screening.

12. Estimated glomerular filtration rate<30mL/min/1.73m ² And/or is undergoing dialysis.

13. Hypersensitive to the test drug (danecopan) or any of its excipients

The statistical method comprises the following steps:

statistically, an improvement in hemoglobin levels at week 12 receiving daidzein Pan Zhiliao relative to baseline compared to improvement in placebo treatment; that is, at week 12, the mean change from baseline was different between danicopan and placebo.

And (3) analyzing the curative effect: changes in baseline and post-baseline measured summary statistics from baseline will be presented by visit to analyze all persistent efficacy variables. Efficacy goals and endpoints are summarized in table 9 below.

TABLE 9 target and endpoint

The primary efficacy endpoint was the change in hemoglobin from baseline at week 12 between danicopan and placebo (defined as the lowest Hgb value, between screening and day 1, including screening and day 1). Longitudinal changes in hemoglobin from baseline were analyzed using a mixed-measures repeat model (MMRM) that included the fixed classification effect of treatment, study visit, and study visit with treatment group interaction, as well as continuous fixed covariates of baseline hemoglobin values and hierarchical randomized indices of transfusion history in the model. The Kenward-Roger approximation will be used to estimate the denominator degrees of freedom. The primary efficacy analysis will be the difference between the dannikopan and placebo groups at week 12 and will be tested.

In addition, a longitudinal image representation will be provided to examine the hemoglobin profile at 12 weeks of treatment with dannikon or placebo plus C5 inhibitor.

The primary efficacy analysis will be based on the ITT population. The primary efficacy endpoints, hemoglobin measurement changes will be support analyzed on a per protocol population basis to examine the impact of primary protocol deviations.

Secondary efficacy analysis will be performed based on the ITT population. The key secondary efficacy endpoints will be analyzed using a tiered fixed sequence test procedure to determine statistical significance.

And (3) safety analysis: all safety analyses were performed in a safe population, including a 12-week blind and a subsequent 12-week open label treatment period. The safety analysis will be based primarily on the frequency of adverse events, clinical laboratory assessments, vital signs and 12-lead electrocardiograms. Other security data will be summarized as appropriate.

Middle-stage analysis: the study trial principal can conduct interim analyses as appropriate (based on feasibility) when about 50% of patients have been randomized into study treatment and have an opportunity to complete a 12-week treatment period of 1 (score of information = 0.5). The purpose of the interim analysis was to assess the efficacy of early discontinuation of the study. Primary endpoints of Hgb level changes at week 12, and key secondary endpoints, if performed, will be evaluated using the alpha consumption method specified below to control family pattern differential rates.

Introduction to

Danicopan (ALXN 2040, formerly ACH-0144471) is a small molecule orally administered Factor D (FD) inhibitor that is currently being developed for the treatment of complement-mediated diseases such as Paroxysmal Nocturnal Hemoglobinuria (PNH) and C3 glomerular disease (C3G). Factor D is a serine protease that catalyzes the cleavage of Factor B (FB), a rate-limiting step in the complement Alternative Pathway (AP). By inhibiting FD, danisco Pan Youxiao and specifically inhibit AP activity. This critical study will evaluate the efficacy and safety of dannikopan in patients with clinically significant extravascular hemolyzation (EVH) of C5 inhibitors (eculizumab or rayleigh mab).

Benefits of

PNH is a serious life-threatening disease and there are some unmet needs for this patient population (approved C5 inhibitors do not address this need), which may be addressed by effective oral FD inhibitors. Three groups of patients have been identified whose PNH is not adequately controlled:

patients with suboptimal responses to eculizumab or rayleigh mab (approximately 30%), probably mainly due to extravascular haemolysis mediated by C3 opsonization. Ecularization of PNH erythrocytes by eculizumab or rayleigh mab therapy without compromising membrane attack complex (end stage of complement pathway); however, it does not prevent the deposition of the C3 fragment on the PNH erythrocyte membrane, which may lead to its extravascular hemolysis. Danicopan has potential mechanical advantage because it acts upstream of C3 cleavage and has been shown to block C3 fragment deposition.

Due to the CR1 gene polymorphisms (e.g., hindIII H/L and L/L genotypes), patients only partially respond to eculizumab or rayleigh mab, which has been hypothesized to result in an increased proportion of C3-opsonized RBCs and may improve the therapeutic response of danicopan.

Rare patients (about 1%) that are unresponsive to eculizumab or rayleigh mab due to a C5 mutation (e.g., arg885 His) may also benefit from darnikopan because it acts on a different target in the complement cascade and should not be affected by the C5 mutation.

Design of research

Overall design

This multi-regional, randomized, double-blind, placebo-controlled, multi-dose, phase 3 study was directed to PNH patients with clinically significant EVH using a C5 inhibitor (eculizumab or rayleigh mab).

Failure of screening

Failure to screen is defined as the patient who consented to participate in the clinical study but subsequently entered the study.

Research medicine

The "study drug" in this protocol refers to danicopan or matched placebo. See table 10.

Table 10: study intervention conducted

Background and concomitant therapies

The use of specific concomitant drugs other than C5 inhibitors will be considered on a case-by-case basis, with the primary investigator and the experimental clients making decisions based on the current knowledge of danicopan and the characteristics of the potential concomitant drug.

Background C5 inhibitor therapy: ekulizumab and Rayleigh lizumab

For data acquisition and analysis purposes, all patients were treated with study drug-conjugated C5 inhibitor therapy (i.e., eculizumab or rayleigh mab). C5 inhibitors used in this manner will be considered background therapy. New drugs used in this manner will also be considered background therapy if the patient switches to a different approved C5 inhibitor after completion of the study at week 24, as the patient needs to take a stable dose and interval of their C5 inhibitor for an extended period of time before the study begins.

The approved dose of C5 inhibitor should not be increased nor shortened by interval during the study (except for the rayleigh mab weight dose based on weight change). If indicated, the dosage of the C5 inhibitor may be reduced and if the dosage is reduced and the patient is unable to tolerate, the dosage may be re-incremented to the previous dosage.

C5 inhibitor therapy will be provided according to local regulations and approval.

In some embodiments, the method will comprise

(eculizumab) was administered intravenously to adult PNH patients at a dose of 600mg per week, four times, followed by 900mg at week 5, and thereafter 900mg every 2 weeks. In pediatrics ( <18 years old) PNH patients will

(eculizumab) intravenous administration: (a) Administering to a subject weighing 40kg and above four times a week at a dose of 900mg, followed by a dose of 1200mg at week 5, and thereafter at a dose of 1200mg every two weeks; (b) Administering to a subject weighing 30kg to less than 40kg twice a week at a dose of 600mg, followed by a dose of 900mg at week 3, and thereafter at a dose of 900mg every two weeks; (c) Administering to a subject weighing 20kg to less than 30kg twice a week at a dose of 600mg, followed by a dose of 600mg at week 3, and thereafter at a dose of 600mg every two weeks; (d) Administering to a subject weighing 10kg to less than 20kg once a week at a dose of 600mg, followed by a dose of 300mg at week 3, and thereafter at a dose of 300mg every two weeks; or (e) to a subject weighing 5kg to less than 10kg once a week at a dose of 300mg, followed by a dose of 300mg at week 2 and thereafter at a dose of 300mg every three weeks.

To reduce the risk of meningococcal infection, all patients were vaccinated with meningococcal infection 3 years or less before starting study drug.

Study of drug dose Change

Based on safety and clinical efficacy, the dose of study drug can be escalated to up to 200mg tid in 50mg increments at defined time points during the initial treatment period and the LTE period using the following criteria. All dose escalations using the laboratory results obtained will be determined by the primary researcher as appropriate after negotiation with the trial principal. This applies for

treatment periods

1 and 2. During the study, the C5 inhibitor + placebo treated group will be incremented in the same manner as the C5 inhibitor + danilapan treated group to maintain blindness.

First dose increment point: if the starting dose is well tolerated and the available safety data are satisfactory, if the patient's Hgb level at week 4 or week 16 has not increased relative to its baseline value (day 1)? 2g/dL or if the patient received a blood transfusion during the first 4 weeks, the patient may be escalated to the next highest dose (to a maximum dose of 200mg tid).

Second dose escalation point: if the patient has not increased Hgb levels at week 8 or week 20? 3g/dL or not normalized from its baseline value (day 1) to at least the midpoint of the normal range for gender, or the patient received a blood transfusion during the first 4 weeks, the patient may be escalated to the next highest dose (maximum dose to 200mg tid).

Third dose escalation point: if the patient's Hgb level at

week

12 or 24 is not normalized to at least the midpoint of the normal range for gender, or the patient received a blood transfusion during the first 4 weeks, the patient may be escalated to the next highest dose (to a maximum dose of 200mg tid).

ALT, AST, GGT and ALP should be measured at the clinic or by visit health care facility blood draws 72 to 96 hours after dose escalation.

Any patient who has not yet been titrated up to 200mg of study drug may be titrated up to 200mg of tid Danicopan if the patient has been titrated for at least 4 weeks with its previous dose and the investigator believes that additional efficacy may be obtained. Dose escalation following week 24 visit will be discussed with the experimental committee prior to implementation. If the patient's dose has been escalated, the dose may be reduced to a lower dose for safety or tolerability reasons after the following negotiations by the investigator and the medical supervisor. The dosage can be re-increased following the same procedure

At any point in time after dose escalation, if patient safety or tolerability warrants reducing the dose to the previous dose, it may be done after negotiation with the trial principal. This dose reduction will only occur if both the primary researcher and the trial principal agree that the patient benefits from the lower dose.

Transfusion guidelines before and during the study when subjects had the following conditions, pRBC transfusion was recommended:

1. hemoglobin values less than 6g/dL, whether or not clinical signs or symptoms are present, or

2. Hemoglobin values of less than 9g/dL are associated with severe signs or symptoms that require transfusion to relieve.

In the event of life-threatening anemia, transfusion of ABO-and RhD-matched blood is appropriate. This additional test is recommended if further matching of Kell and JK antigens can be performed without delaying the provision of blood for emergency transfusion.

Study evaluation and procedure

This section describes the required study assessment procedure. The schedule for all procedures can be found below.

Evaluation of therapeutic effects

Blood will be collected according to an evaluation schedule to evaluate the efficacy endpoints of hemoglobin, reticulocyte counts, bilirubin, and lactate dehydrogenase changes. PNH RBC clone size, C3 fragment deposition on PNH RBCs, AP and CP activity, and Bb, C3 and FD levels will also be assessed. The blood collection procedure is as follows.

Transfusion data from screening to follow-up will be collected including the number of transfused RBC units and associated pre-transfusion hemoglobin values (e.g., reticulocyte count) (from study site records and any other location where the patient received any transfusions) and recorded in the CRF of each patient.

Results reported by the patient

All patients enrolled in the study will self-manage the FACIT fatigue (version 4) Questionnaire, the European Cancer Research and Treatment organization (European organization for Research and Treatment of Cancer (EORTC)) Quality of Life 30 Core Questionnaire (Quality of Life questonnaire-Core 30Scale (QLQ-C30)) Questionnaire, the EQ-5D-3L Questionnaire, and the work efficiency and activity disorder Questionnaire: specific health problem V2.0 (WPAI: SHP, V2.0) (see below). The local language version of each tool will be provided separately.

Medical resource utilization data will be collected according to the schedule shown below. For the HRU, the investigator or prescriber will record the number of outpatient visits, emergency services used, hospital visits, missed work visits for each participant, and record the number of times the patient's urine is darkened.

All PRO and QoL assessments (written or electronic) will be conducted prior to treatment dose, during the planned visit.

Analysis of efficacy

Changes in the baseline and post-baseline measured aggregated statistics from the baseline will be presented by the predefined visit to analyze all sustained efficacy variables.

Analysis of the Primary efficacy

The primary efficacy endpoint was the change in hemoglobin from baseline at week 12 between danicopan and placebo (defined as the lowest Hgb value, between screening and day 1, including screening and day 1). Longitudinal changes in hemoglobin from baseline will be analyzed using a mixed-measures repeat model (MMRM) that includes the fixed classification effect of treatment, study visit and study visit of treatment group interaction, as well as continuous fixed covariates of baseline hemoglobin values and hierarchical randomization indicators of transfusion history in the model. The Kenward-Roger approximation will be used to estimate the denominator degrees of freedom. The primary efficacy analysis will be the comparison between the danicopan and placebo groups at week 12, and the trial will be performed at a bilateral 0.05 level of significance.

The primary objective was to evaluate the effect of danicopan on hemoglobin change after 12 weeks of treatment compared to placebo. To address the effect of transfusions on hemoglobin values, for patients transfused at or after week 8, hemoglobin values collected at week 12 would not be included in the primary efficacy analysis. This rule would also apply to longitudinal observations taken at early visits, i.e. hemoglobin values taken within 4 weeks after transfusion would not be included in the primary efficacy analysis.

Because of the relatively small sample size and the short duration of blind treatment (12 weeks), all efforts were made to minimize the lack of 12-week measurements. In addition, a longitudinal image representation will also be provided to examine the hemoglobin profile within 12 weeks of treatment with danicopan or placebo plus approved C5 inhibitor.

The primary efficacy analysis will be based on the ITT population. The primary efficacy endpoints, hemoglobin measurement changes will be support analyzed on a per protocol population basis to examine the impact of primary protocol deviations. Additional sensitivity analyses will be performed to evaluate the efficacy of the treatment under the assumption of a surrogate missing data mechanism. Such analysis will be detailed in the statistical analysis plan.

Secondary efficacy analysis

Secondary efficacy endpoints are shown below. Secondary efficacy analysis will be performed based on the ITT population.

The key secondary efficacy endpoints (ranked by importance) are as follows. Statistical significance at a 0.05 level was determined for both sides of each endpoint using the hierarchical fixed sequence test procedure.

1. Differences in the proportion of patients who avoided RBC transfusion between danisco Pan Zu and placebo during 12 weeks of treatment

2. Difference in FACIT fatigue score from baseline at week 12 between danac Pan Zu and placebo.

3. Difference in absolute reticulocyte counts from baseline between danecopan and placebo at week 12.

For the ratio parameters, such as patient achieved transfusion avoidance and hemoglobin normalization at week 12, fisher's exact test will be used to compare the danicopan and placebo groups.

For the change in RBC transfusion units/examples from treatment phase 1 12 weeks before treatment initiation, the analysis of covariance (ANCOVA) model (including transfusion units and examples for the treatment group and 12 weeks before treatment initiation) will be used to compare the danicopan and placebo groups.

For changes in numerical endpoints (such as FACIT fatigue score, EQ-5D-3L score, EORTC QLQ-C30 score, absolute reticulocyte count, total bilirubin and direct bilirubin or other PNH-associated biomarkers) at week 12 from baseline, the MMRM model specified in the primary efficacy analysis will be used to compare the mean difference between Danicopan and placebo.

The layered fixed sequence test procedure requires rejection of the current hypothesis; that is, if the p-value of the test statistic is <0.05, then the significance of the next hypothesis is tested starting from the critical secondary endpoint described above according to clinical significance. When the hypothesis cannot be rejected, the sequential testing process will be stopped.

The results of various statistical procedures based on data used to analyze remaining secondary efficacy and exploratory endpoints will be described below:

the number of haemoglobin stabilised patients and the proportion of patients receiving 24 weeks of dannikon treatment during the past 12 weeks of treatment. Hemoglobin stabilization is defined as avoiding a drop in Hgb levels of no more than 0.5g/dL at week 24 from week 12.

Change in RBC transfusion units/cases from 24 weeks prior to initiation of treatment to 24 weeks treatment period for patients receiving 24 weeks of dannikopan treatment.

Proportion of patients who avoid transfusion during the 24-week treatment period, and proportion of patients normalized for hemoglobin at week 24

Change from baseline (day 1) in patients receiving 24 weeks of dannikopan treatment for FACIT fatigue, absolute reticulocyte count, total and direct bilirubin, LDH and other PNH-related biomarkers.

PNH RBC clone size, deposition of C3 fragments on PNH RBCs, measurement of alternative and classical pathway activity, bb, C3 and FD levels.

PRO and QoL endpoints.

Sequence summary

Sequence Listing

<110> Alliexon PHARMACEUTICALS (ALEXION PHARMACEUTICAL, INC.)

<120> use of complement factor D inhibitors alone or in combination with anti-C5 antibodies for the treatment of paroxysmal nocturnal hemoglobinuria

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<151> 2020-06-26

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Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

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Ser Leu Ser Leu Gly Lys

325

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Gly Glu Ile Leu Pro Gly Ser Gly His Thr Glu Tyr Thr Glu Asn Phe

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Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

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Ala Arg Tyr Phe Phe Gly Ser Ser Pro Asn Trp Tyr Phe Asp Val Trp

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Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

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Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr

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Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

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Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys

210 215 220

Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

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Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro

260 265 270

Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

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Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Leu His Glu Ala

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Leu His Ser His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

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Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

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Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Thr Ser Ser Asn Phe Gly Thr Gln Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro

100 105 110

Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

115 120 125

Thr Leu Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp

130 135 140

Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly

145 150 155 160

Met Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn

165 170 175

Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp

180 185 190

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro

195 200 205

Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu

210 215 220

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn

225 230 235 240

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

245 250 255

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

260 265 270

Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

275 280 285

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

290 295 300

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

305 310 315 320

Ser Leu Ser Pro Gly Lys

325

<210> 16

<211> 448

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "artificial sequence description: synthesis of

Polypeptide "

<400> 16

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ile Phe Ser Asn Tyr

20 25 30

Trp Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Glu Ile Leu Pro Gly Ser Gly Ser Thr Glu Tyr Thr Glu Asn Phe

50 55 60

Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Phe Phe Gly Ser Ser Pro Asn Trp Tyr Phe Asp Val Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Thr Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys

210 215 220

Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Thr Arg

245 250 255

Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Met Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val

290 295 300

Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 17

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "artificial sequence description: synthesis of

Peptides "

<400> 17

Gly Ala Ser Glu Asn Ile Tyr His Ala Leu Asn

1 5 10

<210> 18

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "artificial sequence description: synthesis of

Peptides "

<400> 18

Glu Ile Leu Pro Gly Ser Gly His Thr Glu Tyr Thr Glu Asn Phe Lys

1 5 10 15

Asp

<210> 19

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "artificial sequence description: synthesis of

Peptides "

<400> 19

Gly His Ile Phe Ser Asn Tyr Trp Ile Gln

1 5 10

<210> 20

<211> 448

<212> PRT

<213> Artificial sequence

<220>

<221> Source

<223 >/note = "artificial sequence description: synthesis of

Polypeptide "

<400> 20

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly His Ile Phe Ser Asn Tyr

20 25 30

Trp Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Glu Ile Leu Pro Gly Ser Gly His Thr Glu Tyr Thr Glu Asn Phe

50 55 60

Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Phe Phe Gly Ser Ser Pro Asn Trp Tyr Phe Asp Val Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys

210 215 220

Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro

260 265 270

Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

<210> 21

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "artificial sequence description: synthesis of

Peptides "

<400> 21

Ser Tyr Ala Ile Ser

1 5

<210> 22

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "artificial sequence description: synthesis of

Peptides "

<400> 22

Gly Ile Gly Pro Phe Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe Gln

1 5 10 15

Gly

<210> 23

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "artificial sequence description: synthesis of

Peptides "

<400> 23

Asp Thr Pro Tyr Phe Asp Tyr

1 5

<210> 24

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "artificial sequence description: synthesis of

Peptides "

<400> 24

Ser Gly Asp Ser Ile Pro Asn Tyr Tyr Val Tyr

1 5 10

<210> 25

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "artificial sequence description: synthesis of

Peptides "

<400> 25

Asp Asp Ser Asn Arg Pro Ser

1 5

<210> 26

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "artificial sequence description: synthesis of

Peptides "

<400> 26

Gln Ser Phe Asp Ser Ser Leu Asn Ala Glu Val

1 5 10

<210> 27

<211> 116

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "artificial sequence description: synthesis of

Polypeptide "

<400> 27

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr

20 25 30

Ala Ile Ser Val Trp Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Gly Pro Phe Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Thr Pro Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 28

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "artificial sequence description: synthesis of

Polypeptide "

<400> 28

Asp Ile Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln

1 5 10 15

Thr Ala Arg Ile Ser Cys Ser Gly Asp Ser Ile Pro Asn Tyr Tyr Val

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Asp Asp Ser Asn Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Phe Asp Ser Ser Leu Asn Ala

85 90 95

Glu Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 29

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "artificial sequence description: synthesis of

Peptides "

<400> 29

Asn Tyr Ile Ser

1

<210> 30

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "artificial sequence description: synthesis of

Peptides "

<400> 30

Ile Ile Asp Pro Asp Asp Ser Tyr Thr Glu Tyr Ser Pro Ser Phe Gln

1 5 10 15

Gly

<210> 31

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "artificial sequence description: synthesis of

Peptides "

<400> 31

Tyr Glu Tyr Gly Gly Phe Asp Ile

1 5

<210> 32

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "artificial sequence description: synthesis of

Peptides "

<400> 32

Ser Gly Asp Asn Ile Gly Asn Ser Tyr Val His

1 5 10

<210> 33

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "artificial sequence description: synthesis of

Peptides "

<400> 33

Lys Asp Asn Asp Arg Pro Ser

1 5

<210> 34

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "artificial sequence description: synthesis of

Peptides "

<400> 34

Gly Thr Tyr Asp Ile Glu Ser Tyr Val

1 5

<210> 35

<211> 116

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "artificial sequence description: synthesis of

Polypeptides "

<400> 35

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Asn Tyr

20 25 30

Ile Ser Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met Gly

35 40 45

Ile Ile Asp Pro Asp Asp Ser Tyr Thr Glu Tyr Ser Pro Ser Phe Gln

50 55 60

Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr Leu

65 70 75 80

Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys Ala

85 90 95

Arg Tyr Glu Tyr Gly Gly Phe Asp Ile Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 36

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "artificial sequence description: synthesis of

Polypeptide "

<400> 36

Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln

1 5 10 15

Thr Ala Arg Ile Ser Cys Ser Gly Asp Asn Ile Gly Asn Ser Tyr Val

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Lys Asp Asn Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Tyr Asp Ile Glu Ser Tyr Val

85 90 95

Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 37

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> Source

<223 >/note = "artificial sequence description: synthesis of

Peptides "

<400> 37

Ser Ser Tyr Tyr Val Ala

1 5

<210> 38

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "artificial sequence description: synthesis of

Peptides "

<400> 38

Ala Ile Tyr Thr Gly Ser Gly Ala Thr Tyr Lys Ala Ser Trp Ala Lys

1 5 10 15

Gly

<210> 39

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<221> Source

<223 >/note = "artificial sequence description: synthesis of

Peptides "

<400> 39

Asp Gly Gly Tyr Asp Tyr Pro Thr His Ala Met His Tyr

1 5 10

<210> 40

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "artificial sequence description: synthesis of

Peptides "

<400> 40

Gln Ala Ser Gln Asn Ile Gly Ser Ser Leu Ala

1 5 10

<210> 41

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "artificial sequence description: synthesis of

Peptides "

<400> 41

Gly Ala Ser Lys Thr His Ser

1 5

<210> 42

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "artificial sequence description: synthesis of

Peptides "

<400> 42

Gln Ser Thr Lys Val Gly Ser Ser Tyr Gly Asn His

1 5 10

<210> 43

<211> 123

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "artificial sequence description: synthesis of

Polypeptide "

<400> 43

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ser His Ser Ser

20 25 30

Tyr Tyr Val Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp

35 40 45

Val Gly Ala Ile Tyr Thr Gly Ser Gly Ala Thr Tyr Lys Ala Ser Trp

50 55 60

Ala Lys Gly Arg Phe Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val

65 70 75 80

Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ala Ser Asp Gly Gly Tyr Asp Tyr Pro Thr His Ala Met His Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 44

<211> 110

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "artificial sequence description: synthesis of

Polypeptides "

<400> 44

Asp Val Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asn Ile Gly Ser Ser

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Gly Ala Ser Lys Thr His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gln Ser Thr Lys Val Gly Ser Ser

85 90 95

Tyr Gly Asn His Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 45

<211> 451

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "artificial sequence description: synthesis of

Polypeptides "

<400> 45

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val His Ser Ser

20 25 30

Tyr Tyr Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp

35 40 45

Val Gly Ala Ile Phe Thr Gly Ser Gly Ala Glu Tyr Lys Ala Glu Trp

50 55 60

Ala Lys Gly Arg Val Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val

65 70 75 80

Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ala Ser Asp Ala Gly Tyr Asp Tyr Pro Thr His Ala Met His Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

115 120 125

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly

130 135 140

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

145 150 155 160

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

165 170 175

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

180 185 190

Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

195 200 205

Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys

210 215 220

Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

225 230 235 240

Arg Arg Gly Pro Lys Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

245 250 255

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

260 265 270

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

275 280 285

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

290 295 300

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

305 310 315 320

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser

325 330 335

Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

340 345 350

Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln

355 360 365

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

370 375 380

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

385 390 395 400

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

405 410 415

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

420 425 430

Val Leu His Glu Ala Leu His Ala His Tyr Thr Arg Lys Glu Leu Ser

435 440 445

Leu Ser Pro

450

<210> 46

<211> 217

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "artificial sequence description: synthesis of

Polypeptides "

<400> 46

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ser

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Gly Ala Ser Glu Thr Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Asn Thr Lys Val Gly Ser Ser

85 90 95

Tyr Gly Asn Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr

100 105 110

Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu

115 120 125

Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro

130 135 140

Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly

145 150 155 160

Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr

165 170 175

Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His

180 185 190

Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val

195 200 205

Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 47

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "artificial sequence description: synthesis of

Polypeptide "

<400> 47

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Ser Val Ser Ser Ser

20 25 30

Tyr Trp Thr Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Tyr Tyr Ser Gly Ser Ser Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Ala Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Glu Gly Asn Val Asp Thr Thr Met Ile Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 48

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "artificial sequence description: synthesis of

Polypeptide "

<400> 48

Ala Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp

20 25 30

Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ala Gly

50 55 60

Arg Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Asp Phe Asn Tyr Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 49

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "artificial sequence description: synthesis of

Polypeptide "

<400> 49

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Ser Val Ser Ser Ser

20 25 30

Tyr Trp Thr Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Tyr Tyr Ser Gly Ser Ser Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Ala Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Glu Gly Asn Val Asp Thr Thr Met Ile Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro

210 215 220

Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

260 265 270

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

<210> 50

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "artificial sequence description: synthesis of

Polypeptide "

<400> 50

Ala Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp

20 25 30

Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ala Gly

50 55 60

Arg Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Asp Phe Asn Tyr Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 51

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<221> Source

<223 >/note = "artificial sequence description: synthesis of

Polypeptide "

<400> 51

Asp Ile Gln Val Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ile Thr Ser Thr Asp Ile Asp Asp Asp

20 25 30

Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile

35 40 45

Ser Gly Gly Asn Thr Leu Arg Pro Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Leu Gln Ser Asp Ser Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 52

<211> 223

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "artificial sequence description: synthesis of

Polypeptide "

<400> 52

Glu Val Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Thr Tyr Thr Gly Glu Thr Thr Tyr Ala Asp Asp Phe

50 55 60

Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr

65 70 75 80

Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Glu Arg Glu Gly Gly Val Asn Asn Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro

115 120 125

Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val

130 135 140

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala

145 150 155 160

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly

165 170 175

Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly

180 185 190

Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys

195 200 205

Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr

210 215 220

Claims

1. A method for treating Paroxysmal Nocturnal Hemoglobinuria (PNH) in a subject who has previously exhibited an inadequate response to a C5 inhibitor, e.g., an anti-C5 antibody therapy, comprising administering to the subject a therapeutically effective amount of an inhibitor of an alternative component of the complement Alternative Pathway (AP).

2. The method of claim 1, wherein the inhibitor of the surrogate component of AP comprises inhibition of a target upstream of complement 5 (C5), such as factor D or complement 3 (C3).

3. The method of claim 1, wherein upon treatment with the inhibitor of AP, a decrease in one or more of the following is observed in the subject: (a) persistent extravascular hemolysis (EVH); (b) anemia; and/or (c) transfusion-dependent; and/or observing an improvement in the FACIT fatigue scale score of the subject.

4. The method of claim 3, wherein control of MAC-mediated intravascular hemolysis is maintained or improved in the insufficiently responsive PNH subject following treatment with the inhibitor of AP.

5. The method of claim 1 or 2, wherein an inadequate response to anti-C5 antibody therapy is associated with: (I) Pharmacokinetic (PK) aspects, e.g., (a) ineffective inhibition of C5 cleavage in the subject; (b) Low dose of the anti-C5 antibody and/or low subject plasma levels; (C) enhanced clearance of anti-C5 antibodies in the subject; or (d) anti-C5 antibody intolerance in the subject results in a reduction in anti-C5 antibody dose, preferably wherein anti-C5 antibody intolerance comprises fatigue and post-infusion pain; or (II) Pharmacodynamic (PD) aspects, e.g., (a) CR1 polymorphism; (b) Extravascular hemolysis (EVH), e.g., opsonization of surviving blood cells by intravascular hemolysis (IVH); and (C) the deleterious effects of the C3 fragment on the activity of anti-C5 antibodies.

6. A method for treating Paroxysmal Nocturnal Hemoglobinuria (PNH) in a subject who previously exhibited an inadequate response to anti-C5 antibody therapy, the method comprising:

(a) An increase in hemoglobin of 2.0g/dL or higher compared to the subject's baseline hemoglobin level;

(b) Transfusion independence; and/or

(c) A chronic disease treatment Function Assessment (FACIT) fatigue scale score is increased by 10 points or more compared to the subject's baseline FACIT fatigue scale score.

7. A method for treating Paroxysmal Nocturnal Hemoglobinuria (PNH) in a subject who previously exhibited an inadequate response to anti-C5 antibody therapy, the method comprising:

wherein the subject's inadequate response is transfusion-dependent and/or anemia; and is provided with

(b) Transfusion independence; and/or

8. A method for treating Paroxysmal Nocturnal Hemoglobinuria (PNH) in a subject, the method comprising:

(a) An increase in hemoglobin of 2.0g/dL or more compared to the baseline hemoglobin level of the subject;

(b) Transfusion independence; and/or

9. The method of claim 6 or 7, wherein the subject previously exhibited an inadequate response to eculizumab.

10. The method of claim 9, wherein the subject has previously received an approved dose or higher of eculizumab for ≧ 24 weeks without alteration of the regimen for ≦ 8 weeks.

11. The method according to claim 7 or 8, wherein the anti-C5 antibody or antigen-binding fragment thereof administered to the subject is a human antibody humanized antibody, bispecific antibody, chimeric antibody, fab'2, scFv, SMIP, and,

Nanobodies or C5-inhibiting domain antibodies.

12. The method of claim 7 or 8, wherein the anti-C5 antibody or antigen-binding fragment thereof administered to the subject comprises CDR1, CDR2, and CDR3 heavy chain sequences as set forth in SEQ ID NOS 1, 2, and 3, respectively, and CDR1, CDR2, and CDR3 light chain sequences as set forth in SEQ ID NOS 4, 5, and 6, respectively.

13. The method of claim 7-8 or 12, wherein the anti-C5 antibody or antigen-binding fragment thereof administered to the subject comprises: the heavy chain variable region comprising SEQ ID NO 7 and the light chain variable region comprising SEQ ID NO 8.

14. The method of claims 7-8 or 12-13, wherein the anti-C5 antibody or antigen-binding fragment thereof administered to the subject comprises: a heavy chain comprising SEQ ID NO 10 and a light chain comprising SEQ ID NO 11.

15. The method of claims 7-8 or 12-14, wherein the anti-C5 antibody administered to the subject is

16. The method of claim 7 or 8, wherein the anti-C5 antibody or antigen-binding fragment thereof administered to the subject comprises CDR1, CDR2, and CDR3 heavy chain sequences as set forth in SEQ ID NOS 19, 18, and 3, respectively, and CDR1, CDR2, and CDR3 light chain sequences as set forth in SEQ ID NOS 4, 5, and 6, respectively.

17. The method of claim 7-8 or 16, wherein the anti-C5 antibody or antigen-binding fragment thereof administered to the subject further comprises a variant human Fc constant region that binds human neonatal Fc receptor (FcRn), wherein the variant human Fc CH3 constant region comprises Met-429-Leu and Asn-435-Ser substitutions at residues corresponding to methionine 428 and asparagine 434 of a native human IgG Fc constant region, each numbered in the EU.

18. The method of claims 7-8 or 16-17, wherein the anti-C5 antibody or antigen-binding fragment thereof administered to the subject comprises: the heavy chain variable region comprising SEQ ID NO 12 and the light chain variable region comprising SEQ ID NO 8.

19. The method of claims 7-8 or 16-18, wherein the anti-C5 antibody or antigen-binding fragment thereof administered to the subject further comprises: the heavy chain constant region depicted in SEQ ID NO 13.

20. The method of claims 7-8 or 16-19, wherein the anti-C5 antibody or antigen-binding fragment thereof administered to the subject comprises: a heavy chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO. 14 and a light chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO. 11.

21. The method of claims 7-8 or 16-20, wherein the anti-C5 antibody administered to the subject is rayleigh mab.

22. The method of any one of the preceding claims, wherein the CFD inhibitor is a small molecule inhibitor, a nucleotide, a peptide, a protein, a peptide mimetic, an aptamer, or any other molecule that binds to factor D.

23. The method of any one of the preceding claims, wherein the CFD inhibitor is a nucleotide selected from the group consisting of: DNA, RNA, shRNA, miRNA, siRNA, antisense DNA.

24. The method of any one of the preceding claims, wherein the CFD inhibitor is an antibody or antigen-binding fragment thereof that binds factor D.

25. The method of any one of claims 6-22, wherein the CFD inhibitor comprises:

or a pharmaceutically acceptable salt thereof.

26. The method of any one of claims 6-22 and 25, wherein the CFD inhibitor is danicopan.

27. The method of any one of claims 6-22 and 25-26, wherein the CFD inhibitor is administered orally to the subject.

28. The method of any one of claims 6-22 and 25-27, wherein the CFD inhibitor is orally administered to the subject three times daily (TID).

29. The method of any one of claims 6-22 and 25-28, wherein the CFD inhibitor is orally administered to the subject at a dose TID of 100mg, 150mg, or 200 mg.

30. The method of any one of the preceding claims, wherein the CFD is administered for 24 weeks.

31. The method of any one of the preceding claims, wherein the CDR is administered for 9 months, 12 months, 15 months, 20 months, 24 months, or longer.

32. The method of any one of claims 7-31, wherein the anti-C5 antibody or antigen-binding fragment is administered intravenously to the subject.

33. The method of any one of claims 7-15 and 22-32, wherein the anti-C5 antibody or antigen-binding fragment thereof is administered to the subject at a dose of 600mg per week, four times, followed by 900mg at week 5, and thereafter 900mg every 2 weeks.

34. The method of any one of claims 7-15 and 22-32, wherein the subject is less than 18 years of age.

35. The method of claim 34, wherein the anti-C5 antibody or antigen-binding fragment thereof is administered to the subject younger than 18 years:

(e) Subjects weighing 5kg to less than 10kg were administered once a week at a dose of 300mg, followed by a dose of 300mg on week 2 and thereafter at a dose of 300mg every three weeks.

36. The method of any one of claims 7-11 and 16-32, wherein the anti-C5 antibody or antigen-binding fragment thereof is administered as follows:

37. The method of any one of claims 7-11 and 16-32, wherein the subject is less than 18 years of age.

38. The method of claim 37, wherein the anti-C5 antibody or antigen-binding fragment thereof is administered as follows:

(b) On day 15 and every four weeks thereafter, the doses were: 300mg of patients with the weight of more than or equal to 5 to less than 10kg or 600mg of patients with the weight of more than or equal to 10 to less than 20 kg; or on day 15 and every eight weeks thereafter, the doses were: 2100mg of patients with a weight of more than or equal to 20kg to less than 30kg, 2700mg of patients with a weight of more than or equal to 30kg to less than 40kg, 3000mg of patients with a weight of more than or equal to 40kg to less than 60kg, 3300mg of patients with a weight of more than or equal to 60kg to less than 100kg, or 3600mg of patients with a weight of more than or equal to 100 kg.

39. The method of any one of claims 7-37, wherein the anti-C5 antibody or antigen-binding fragment thereof is administered to the subject for 12 or 24 weeks.

40. The method of any one of claims 7-37, wherein the anti-C5 antibody or antigen-binding fragment thereof is administered to the subject for 9 months, 12 months, 15 months, 20 months, 24 months, or longer.

41. The method of any one of the preceding claims, wherein the treatment shifts bilirubin to normal levels.

42. The method of any one of the preceding claims, wherein the treatment results in a reduction of reticulocytes as compared to baseline.

43. The method of any one of the preceding claims, wherein the treatment results in an increase in PNH-specific erythrocyte clone size compared to baseline.

44. The method of any one of the preceding claims, wherein the treatment results in a reduction of PNH red blood cells opsonized with the C3 fragment as compared to baseline.

45. The method of any one of the preceding claims, wherein the treatment results in reduced hemolysis as assessed by Lactate Dehydrogenase (LDH) levels compared to baseline.

46. The method of any one of the preceding claims, wherein the treatment reduces the need for transfusion compared to baseline.

47. The method of any one of the preceding claims, wherein the treatment results in terminal complement inhibition.

48. The method of any one of the preceding claims, wherein the treatment produces at least one therapeutic effect selected from the group consisting of: abdominal pain, dyspnea, dysphagia, chest pain, and erectile dysfunction are reduced or halted compared to baseline.

49. The method of any one of the preceding claims, wherein the treatment shifts to normal levels at least one or more hemolysis-related blood biomarker selected from the group consisting of: free hemoglobin, haptoglobin, reticulocyte count, PNH Red Blood Cell (RBC) clone, and/or D-dimer.

50. The method of any one of the preceding claims, wherein the treatment reduces Major Adverse Vascular Events (MAVEs).

51. The method of any one of the preceding claims, wherein the treatment shifts estimated glomerular filtration rate (eGFR) or point of care urine albumin creatinine and plasma Brain Natriuretic Peptide (BNP) to normal levels.

52. The method of any one of the preceding claims, wherein the treatment alters quality of life relative to baseline as assessed by the quality of life 30 core questionnaire scale of 4 th edition and european cancer research and treatment organizations.

53. The method of any one of the preceding claims, comprising determining the subject's hemoglobin level, transfusion status, and/or FACIT fatigue scale score at baseline and 12 or 24 weeks post-treatment, wherein

(b) Transfusion independence; and/or

(c) Increasing the FACIT fatigue scale score by 10 points or more compared to the subject's baseline FACIT fatigue scale score

Is indicative of treatment.

54. A method for treating Paroxysmal Nocturnal Hemoglobinuria (PNH) in a subject who has previously responded inadequately to treatment with eculizumab, the method comprising:

administering to the subject a therapeutically effective amount of danicopan in combination with a therapeutically effective amount of eculizumab,

wherein the subject's inadequate response is transfusion-dependent and/or anemia; and is

wherein eculizumab is administered to the subject intravenously at a dose of 600mg per week, four times, followed by 900mg at week 5, and thereafter 900mg every 2 weeks; and is

Wherein the subject exhibits one or more of the following clinical improvements 12 or 24 weeks after treatment with the CFD inhibitor:

(b) Transfusion independence; and/or

55. A method for treating Paroxysmal Nocturnal Hemoglobinuria (PNH) in a subject who has previously responded inadequately to treatment with eculizumab, the method comprising: administering to the subject a therapeutically effective amount of danicopan in combination with a therapeutically effective amount of eculizumab,

wherein eculizumab is administered intravenously to a subject less than 18 years of age:

i. an increase in hemoglobin of 2.0g/dL or higher compared to the subject's baseline hemoglobin level;

transfusion independent; and/or

56. A method for treating Paroxysmal Nocturnal Hemoglobinuria (PNH) in a subject, the method comprising:

wherein eculizumab is administered to the subject intravenously at a dose of 600mg per week, four times, followed by a dose of 900mg at week 5, and thereafter a dose of 900mg every 2 weeks; and is

(b) Transfusion independence; and/or

57. A method for treating Paroxysmal Nocturnal Hemoglobinuria (PNH) in a subject less than 18 years of age, the method comprising: administering to the subject a therapeutically effective amount of danicopan in combination with a therapeutically effective amount of eculizumab,

wherein eculizumab is administered intravenously:

transfusion independent; and/or

58. The method of any one of the preceding claims, wherein the inhibitor of an alternative component of the complement Alternative Pathway (AP) is selected from the group consisting of:

b) Factor D (FD) inhibitors (e.g., anti-FD mabs such as laparizumab; or small molecule FD inhibitors such as darnikopan (ACH-4471) or BCX 9930);

c) Factor B inhibitors (e.g., LNP 023);

d) Compstatin molecules or derivatives thereof (e.g., APL2, APL9, AMY-101);

e) Mini factor H (e.g., mini FH AMY-201); and

f) Factor H fusion proteins (e.g., TT 30).

59. The method of any one of the preceding claims, wherein the C5 inhibitor is selected from the group consisting of:

a) Eculizumab biosimilar (e.g., ABP 959; elizaria; SB 12);

b) Nomacopan (Coversin; rVA 576);

c) Rayleigh mab;

d) Texazumab (LFG 316);

e) Parse Li Shankang; and

f) Kovar Li Shankang (SKY 059).

60. The method of any one of the preceding claims, wherein the complement replacement pathway (AP) comprises a pharmaceutical composition comprising danicopan.

61. The method of any one of the preceding claims, comprising: administering to the human subject a pharmaceutical composition comprising about 100 to about 200mg of dannikopan every 8 hours.

62. The method of any one of the preceding claims, wherein the subject exhibits extravascular hemolysis (EVH) prior to treatment.

63. A kit for treating Paroxysmal Nocturnal Hemoglobinuria (PNH) in a subject, the kit comprising:

(a) A dose of Complement Factor D (CFD) inhibitor; and

(b) Instructions for using the CFD in a method as claimed in any one of the preceding claims.

64. A kit for treating Paroxysmal Nocturnal Hemoglobinuria (PNH) in a subject, the kit comprising:

(a) A dose of Complement Factor D (CFD) inhibitor;

(b) Dose of anti-C5 antibody; and

(c) Instructions for using the CFD and anti-C5 antibody in the method of any one of the preceding claims.

65. The kit of claim 63 or 64, wherein the CFD is Danisco.

66. The kit of claim 64 or 65, wherein the anti-C5 antibody is eculizumab.

67. The kit of claim 64 or 65, wherein the anti-C5 antibody is Rayleigh mab.

68. A method of treating clinically significant extravascular hemolysis (EVH) in a patient having Paroxysmal Nocturnal Hemoglobinuria (PNH) who has previously been treated with a C5 inhibitor, e.g., an anti-C5 antibody therapy, comprising administering to the subject a therapeutically effective amount of an inhibitor of an alternative component of the complement Alternative Pathway (AP).

69. The method of claim 68, wherein the inhibitor of the surrogate component of AP comprises inhibition of a target upstream of complement 5 (C5), such as factor D or complement 3 (C3).

70. The method of claim 69, wherein the inhibitor of a factor D inhibitor comprises danilaplon.

71. The method of any one of claims 68-70, wherein the clinically significant EVH comprises (a) anemia (e.g., hgb ≦ 9.5 g/dL) and absolute reticulocyte count ≧ 120 x 10 ⁹ L; and/or (b) at least 1 packed RBC or whole blood infusion within 6 months prior to treatment with the inhibitor of the surrogate component of complement AP.

72. The method of any one of claims 68-71, wherein the treatment avoids Transfusion (TA) in PNH patients with clinically significant EVH.

73. The method of claim 72, wherein a treated PNH patient with clinically significant EVH does not require pRBC transfusion, e.g., the patient requires pRBC transfusion when: (1) Hemoglobin values less than 6g/dL, regardless of the presence or absence of clinical signs or symptoms of PNH; or (2) hemoglobin values <9g/dL, with severe signs or symptoms that require transfusion for remission.

74. The method of any one of claims 68-73, wherein the therapeutically effective amount of the inhibitor of the surrogate component of AP complement comprises a daily dose of 600mg of danekang.

75. The method of any one of claims 68-74, wherein the patient is treated with an anti-C5 antibody and a therapeutically effective amount of an inhibitor of the surrogate component of AP complement.

76. The method of claim 75, wherein the anti-C5 antibody therapy comprises therapy with eculizumab (e.g., a pharmaceutical composition comprising eculizumab) or Raylelizumab (e.g., a pharmaceutical composition comprising Raylelizumab) at a standard dose and/or dosing regimen for each antibody in PNH therapy.